Strong activation domain

ABSTRACT

A new and strong transcriptional activation domain was identified from the  Arabidopsis  protein Ethylene Response Factor 98 (AtERF98). This domain has been designated as the “EDLL domain” and has a number of highly conserved amino acid residues that are found throughout the members of the AtERF98 family from plants, including in monocot and eudicot orthologs. The EDLL domain was shown to be highly active when it was fused to transcription factors from plant and yeast, and was also shown to have activation potential comparable to the widely-used VP16 activation domain derived from Herpes simplex. The EDLL domain was also active when it was targeted to a gene promoter by a sequence-specific DNA binding protein or by protein-protein interactions. Unlike other known activation domains such as VP16 and GAL4, the EDLL domain is relatively small in size, and being of plant origin, it is favored as a strong transcriptional activation tool for application in transgenic food crops.

This application is a continuation application of U.S. application Ser. No. 13/000,488, filed on Feb. 4, 2011 (pending), which is the 35 U.S.C. 371 National Stage of International Application No. PCT/US09/048814, filed Jun. 26, 2009 (expired), which claims the benefit of U.S. Provisional Application No. 61/076,534, filed Jun. 27, 2008 (expired). This application is a continuation-in-part of U.S. application Ser. No. 12/705,845, filed on 15 Feb. 2010 (pending), which is a continuation-in-part of U.S. application Ser. No. 10/714,887, filed on Nov. 13, 2003 (now abandoned). U.S. application Ser. No. 12/705,845 is also a continuation-in-part of U.S. application Ser. No. 11/981,576, filed Oct. 30, 2007, which issued as U.S. Pat. No. 7,888,558 on Feb. 15, 2011. This application is also a continuation-in-part of U.S. application Ser. No. 10/903,236, filed on Jul. 30, 2004 (pending). All the above-referenced applications are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to plant genomics and plant improvement, and modifying gene and protein expression.

BACKGROUND OF THE INVENTION

A transcriptional activation domain (TAD) is the region of a transcription factor (TF) protein that is necessary for its transcriptional activation activity when bound to a promoter. The TAD can be present at any location in the protein. These sequences are usually transportable, that is, they retain activation function when isolated from their native proteins and fused to any sequence specific DNA binding domain (DBD) protein. Hence, a TAD:DBD fusion can be used to turn on the expression of any desired target gene, when the promoter of that target gene contains a specific DNA sequence bound by the DBD. This property of TADs extends their utility in various agriculture and medicinal research. TADs are routinely being used in the study of protein-protein and protein-DNA interactions, and also being used for the targeted induction of genes in plants, animals and yeast.

TADs can be classified into three major classes depending upon their amino acid composition: proline-rich, glutamine-rich and acidic-rich. Most well characterized TADs, which confer strong transcriptional activation potential, including the yeast activator protein GAL4 and the VP16 protein from herpes simplex virus, fall in the category of acidic activators. These activation domains, though they are typically large in size, are routinely used for inducing gene expression, and for, protein-protein and protein-DNA interaction studies in yeast, plants and other animal science research.

The acidic activators form an amphipathic structure, that is, the activation domain contains many acidic and polar amino acids residues interspersed with hydrophobic residues. Such stretches of acidic amino acids are widely distributed in various proteins, but all regions rich in acidic amino acids do not necessarily have role in activation. Due to the loose consensus in the amino acid sequence conservation among activators, it is difficult to predict whether or not a given protein sequence has a role in transcriptional activation.

Activation domains that presently used in the art are generally derived from non-plant proteins such as GALA protein (yeast) and VP16 viral protein (herpes simplex virus). Due to their large size, fusion of these domains to a TF can lead to a change in the native structure which compromises the function of that TF. In addition, it may be considered undesirable to use sequences from non-plant proteins in plants destined for commercial use as transgenic crops, particularly those grown for food purposes.

SUMMARY OF THE INVENTION

The EDLL domain is a new activation domain identified from a plant protein. It is highly active when fused with different classes of proteins from plants and yeast, and has activation potential comparable to the widely used VP16 activation domain. Unlike other known strong activation domains such as VP16 and GAL4, EDLL is relatively small in size; fusion of such a small peptide to any protein has a lower chance of altering the native conformation of the fusion protein. The EDLL domain is also present in many plant species, including useful crop species such as rice, maize, soybean and alfalfa. The EDLL domain from these crops or from other plant species can be fused with transcription factors isolated from the same species, or other plant species, and can be used for enhanced induction of any target genes in those crop varieties. This approach affords enhanced activation of TF targets while avoiding contamination of the crop genome with expressed genetic materials derived from outside of the plant kingdom.

The invention thus pertains to a chimeric polypeptide that may be used to increase the expression of a polynucleotide sequence in a host cell or plant. The chimeric polypeptide comprises a transcription activation domain that is covalently fused to a transcription regulatory polypeptide, containing a DBD. The transcription activation domain generally comprises the consensus sequence EX₄DX₃LX₃L (SEQ ID NO: 55), or the consensus sequence E-L/F-X₂₋L/F-D-D/N-X₂-L-X₂-L/M-L (SEQ ID NO: 56), or the consensus sequence E-F/L-X-X-L/F-D-D/N-X-V/L/I-L-X-X-L/M-L (SEQ ID NO: 94), or the consensus sequence E-F/L-E/V-Y/C/F-L/F-D-D/N-X-V/L-L-E/Q/D-E/D/S-L/M-L (SEQ ID NO: 95).

Specific examples of activation domains described by the consensus sequence SEQ ID NOs: 55, 56, 94 or 95 are provided. The transcription activation domain and the transcription regulatory protein within the chimeric polypeptide do not occur in nature in the same polypeptide, or do not occur in nature with the same order or orientation or with the same spacing within the same peptide, that is, they are mutually heterologous. The transcription activation domain and the transcription regulatory protein in the chimeric polypeptide also do not occur in the same copy number or configuration in nature.

The chimeric polypeptide is able to activate the transcription of a target polynucleotide sequence to which the chimeric polypeptide binds.

The invention also pertains to a nucleic acid construct encoding a chimeric polypeptide, as described in the preceding paragraph, that may be used to increase the expression of a polynucleotide sequence after introducing the nucleic acid construct into a host cell.

The invention is also directed to host cells and transgenic plants that are transformed with the nucleic acid construct described in the preceding paragraph.

The invention is also directed to a method for increasing the expression of a polynucleotide sequence in a host cell by introducing the nucleic acid construct described above into the host cell.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND DRAWINGS

The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the invention. The traits associated with the use of the sequences are included in the Examples.

Incorporation of the Sequence Listing

The copy of the Sequence Listing being submitted electronically with this patent application, provided under 37 CFR § 1.821-1.825, is a read-only memory computer-readable file in ASCII text format. The Sequence Listing is named “MBI-0084CON_ST25.txt”, the electronic file of the Sequence Listing was created on Nov. 8, 2013, and is 123 kilobytes in size (measured in MS-WINDOWS). The Sequence Listing is herein incorporated by reference in its entirety.

FIG. 1 shows an optimal alignment of the conserved “EDLL” activation domain found in AP2 transcription factors orthologous to the Arabidopsis AtERF98 (G1792) protein (these proteins and other phylogenetically- and closely-related sequences constitute the “G1792 clade”). Functional G1792 clade members contain, at relative positions, a glutamic acid residue at position 3, an aspartic acid residue at position 8, and a leucine residue at positions 12 and 16. FIG. 1 also provides a sequence logo of the EDLL domain, which consists of stacks of symbols, one stack for each position in the sequence. The overall height of the stack at any position indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino or nucleic acid at that position (see Schneider et al. (1990) Nucleic Acids Res. 18: 6097-6100; Crooks et al. (2004) Genome Res. 14: 1188-1190; or weblogo.berkeley.edu). This sequence logo thus provides a graphical representation of the relative frequencies of the amino acids found in this alignment and in the Sequence listing in the EDLL consensus sequence SEQ ID NO: 95.

FIG. 2 illustrates the results of experiments to demonstrate experimentally the function of the EDLL motif. A 24 amino acid motif comprising the EDLL domain (SEQ ID NO: 37) of AtERF98 (G1792; SEQ ID NO: 2) was fused with a sequence-specific GAL4 DNA binding domain (DBD; encoded by SEQ ID NO: 117) from yeast (GAL4 DBD or “GD”). The chimeric protein, (“GD-EDLL” in this figure; (SEQ ID NO: 118), when expressed in plant protoplasts, induced the expression of a GUS (β-glucuronidase) reporter gene containing GAL4 DBD binding sequences in the promoter (the GUS reporter system makes use of the fluorescent β-glucuronidase substrate, 4-methylumbelliferyl beta-D-glucuronide (MUG), to determine the expression level of the GUS gene). The GAL4 DBD without the EDLL motif (“GD” in this figure; encoded by SEQ ID NO: 117) could not induce the expression of the reporter gene significantly. The activation of the reporter gene by EDLL motif is comparable in magnitude to that obtained with the widely used VP16 activation domain from herpes simplex virus (comparing “GD-VP16”, encoded by SEQ ID NO: 122, and GD-EDLL, encoded by SEQ ID NO: 118, in this figure). When the conserved hydrophobic leucine residues were changed to valine (“EDLLm”; encoded by SEQ ID NO: 119), the activation potential of EDLL motif was significantly compromised.

FIG. 3 shows a graph of relative functional activity determined by either fusing one or two copies of the AtERF98 (G1792) EDLL domain (SEQ ID NO: 37) to a sequence-specific GAL4 DNA binding domain from yeast (encoded by SEQ ID NO: 117), and co-expressing these constructs in plant protoplasts with a reporter construct comprising GAL4-UAS fused to a GUS sequence (as described above for FIG. 2). When either one copy [GD:EDLL(1×)] or two copies [GD:EDLL(2×)] of the AtERF98 EDLL motif are fused to the GAL4 DNA binding domain, reporter gene activity was significantly higher than with the GAL4 DNA binding domain alone (GD), and comparable to the activity obtained with a VP16 activation domain (GD:VP16).

The results provided in FIG. 4 demonstrate that the EDLL motif confers transcriptional activation function to a plant sequence specific DNA binding transcription factor, specifically NF-YB1 (G481, SEQ ID NO: 73). NF-Y (Nuclear Factor-Y) proteins, also referred to as CCAAT sequence binding proteins, consist of three subunits; NF-YA, NF-YB, and NF-YC, all of which are necessary for DNA binding. NF-YB proteins interact with NF-YC proteins as part of a heterotrimeric DNA binding complex (the NF-YB/NF-YC heterodimer is translocated into the nucleus, the NF-YA subunit interacts with the NF-YB:NF-YC heterodimer, and the resulting complex is able to recognize and bind to a “CCAAT” penta-nucleotide element), and this interaction can be detected in plant protoplasts in a two-hybrid assay, with one protein fused to an activation domain and another fused to a DNA binding domain. To demonstrate the utility of the EDLL domain in activating transcription when fused to a heterologous transcription factor, the EDLL motif of AtERF98 was fused to G481 (an NF-YB subunit; SEQ ID NO: 96), and the yeast GAL4 DNA binding domain (GD; encoded by SEQ ID NO: 117) was fused to G483 (SEQ ID NO: 74; an NF-YC subunit). When the GD:G483 chimeric protein (encoded by SEQ ID NO: 121) was expressed in plant protoplasts along with a reporter gene containing GAL4 binding sequences, the GD-G483 chimeric protein alone could not induce reporter gene activity. When G481 (SEQ ID NO: 73) was co-expressed without an EDLL fusion and GD-G483 in protoplasts, the G481+G483 dimer could also not induce the activity of the reporter gene. This indicated that the NF-YB/NF-YC dimer alone is not sufficient to induce the reporter gene activity. When the G481:EDLL fusion (encoded by SEQ ID NO: 96) was co-expressed with GD:G483 in the protoplasts, the G481: EDLL/GD:G483 dimer induced the activity of reporter gene to a significant degree. This interaction was specific to the dimerization of G481 and G483, because the G481:EDLL fusion did not activate the reporter gene when co-expressed with the GD alone (GD+G481:EDLL). The GAL4 DNA binding domain fused directly to the VP16 activation domain (GD:VP16; SEQ ID NO: 122) served as a positive control for activation. A similar experiment was conducted with another NF-YC protein, G715, SEQ ID NO: 75, and the result was similar to that with G483 (shown in figure). This indicated that the EDLL motif can function in larger complexes, and can confer transcriptional activation function to a plant transcription factor lacking strong activation capacity. It is also active even if the protein is not binding DNA directly (G481:EDLL alone can not bind DNA; data not shown) but is recruited to the DNA via interaction with another DNA binding protein (GD:G483 or GD:G715).

FIG. 5 demonstrates that addition of the EDLL domain to a transcriptional repressor can convert it to a transcriptional activator. G400 (SEQ ID NO: 116) is a homeodomain-leucine zipper (HD-Zip) transcription factor that contains a repression domain termed an EAR domain (Ciarbelli et al. (2008) Plant Mol Biol. 68: 465-478). This protein binds to the promoter of another HD-Zip gene (prG398; SEQ ID NO: 99), but does not activate transcription (Myc:G400; encoded by SEQ ID NO: 128) relative to a non-specific control construct (CAT). Addition of the EDLL domain to this transcription factor (G400:EDLL:Myc; encoded by SEQ ID NO: 97) produced significant activation of prG398:GUS fusion construct (SEQ ID NO: 99:GUS), even though the native repression domain was still present. Addition of the EDLL domain to a variant of G400 with the EAR domain mutated (G400EAR:EDLL:Myc; encoded by SEQ ID NO: 98) produced greater activation of the reporter fusion. These results demonstrate that addition of the EDLL domain to a transcription factor with transcriptional repression activity can at least partially overcome the effect of the repression domain.

FIG. 6 demonstrates the utility of EDLL domains from other plant species. When fused to the GAL4 DNA binding domain (GD; encoded by SEQ ID NO: 117), EDLL domains from A. thaliana (GD:G30EDLL, encoded by SEQ ID NO: 123; GD:G1792EDLL, encoded by SEQ ID NO: 117 fused to SEQ ID NO: 37), soy (GD:G3518EDLL, encoded by SEQ ID NO: 124), M. truncatula (GD:G3735EDLL, encoded by SEQ ID NO: 125), rice (GD:G3737EDLL, encoded by SEQ ID NO: 126) and maize (GD:G3739EDLL, encoded by SEQ ID NO: 127) all produced significant transcriptional activation of a chimeric reporter gene containing GAL4 DBD binding sequences in the promoter.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to polynucleotides and polypeptides for modifying phenotypes of plants, particularly those associated with increased abiotic stress tolerance, increased biotic stress tolerance and increased yield with respect to a control plant (for example, a wild-type plant, a non-transformed plant, or a plant transformed with an “empty” nucleic acid construct lacking a polynucleotide of interest comprised within a nucleic acid construct introduced into an experimental plant). Throughout this disclosure, various information sources are referred to and/or are specifically incorporated. The information sources include scientific journal articles, patent documents, textbooks, and World Wide Web browser-inactive page addresses. While the reference to these information sources clearly indicates that they can be used by one of skill in the art, each and every one of the information sources cited herein are specifically incorporated in their entirety, whether or not a specific mention of “incorporation by reference” is noted. The contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the invention.

As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality of such host cells, and a reference to “a stress” is a reference to one or more stresses and equivalents thereof known to those skilled in the art, and so forth.

Definitions

“Polynucleotide” is a nucleic acid molecule comprising a plurality of polymerized nucleotides, for example, at least about 15 consecutive polymerized nucleotides. A polynucleotide may be a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5′ or 3′ untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single-stranded or double-stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, for example, genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can be combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA). The polynucleotide can comprise a sequence in either sense or antisense orientations. “Oligonucleotide” is substantially equivalent to the terms amplimer, primer, oligomer, element, target, and probe and is preferably single-stranded.

A “recombinant polynucleotide” is a polynucleotide that is not in its native state, for example, the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, for example, separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity. For example, the sequence at issue can be cloned into a nucleic acid construct, or otherwise recombined with one or more additional nucleic acids.

An “isolated polynucleotide” is a polynucleotide, whether naturally occurring or recombinant, that is present outside the cell in which it is typically found in nature, whether purified or not. Optionally, an isolated polynucleotide is subject to one or more enrichment or purification procedures, for example, cell lysis, extraction, centrifugation, precipitation, or the like.

“Gene” or “gene sequence” refers to the partial or complete coding sequence of a gene, its complement, and its 5′ or 3′ untranslated regions. A gene is also a functional unit of inheritance, and in physical terms is a particular segment or sequence of nucleotides along a molecule of DNA (or RNA, in the case of RNA viruses) involved in producing a polypeptide chain. The latter may be subjected to subsequent processing such as chemical modification or folding to obtain a functional protein or polypeptide. A gene may be isolated, partially isolated, or found with an organism's genome. By way of example, a transcription factor gene encodes a transcription factor polypeptide, which may be functional or require processing to function as an initiator of transcription.

Operationally, genes may be defined by the cis-trans test, a genetic test that determines whether two mutations occur in the same gene and that may be used to determine the limits of the genetically active unit (Rieger et al. (1976) Glossary of Genetics and Cytogenetics: Classical and Molecular, 4th ed., Springer Verlag, Berlin). A gene generally includes regions preceding (“leaders”; upstream) and following (“trailers”; downstream) the coding region. A gene may also include intervening, non-coding sequences, referred to as “introns”, located between individual coding segments, referred to as “exons”. Most genes have an associated promoter region, a regulatory sequence 5′ of the transcription initiation codon (there are some genes that do not have an identifiable promoter). The function of a gene may also be regulated by enhancers, operators, and other regulatory elements.

The terms “chimeric”, “fusion” and “composite” are used to denote a protein, peptide domain or nucleotide sequence or molecule containing at least two component portions which are mutually heterologous in the sense that they are not, otherwise, found directly (covalently) linked in nature. That is, the component portions are not found in the same continuous polypeptide or gene in nature, at least not in the same copy number, order, configuration or orientation or with the same spacing present in the chimeric protein or composite domain. Specifically, the chimeric polypeptides comprised herein each comprise a transcription regulatory protein and a transcription activation domain that are derived from different sources, or may be present in a different copy number, or may be present in a different configuration, than is found in nature.

Such materials contain components derived from at least two different proteins or genes or from at least two non-adjacent portions of the same protein or gene. Composite proteins, and DNA sequences which encode them, are recombinant in the sense that they contain at least two constituent portions which are not otherwise found directly linked (covalently) together in nature.

“Heterologous” with respect to polynucleotide or polypeptide sequences refers to sequences that are of different origins, such as, for example, from different organisms, different genes or proteins, different regions of a chromosome, different chromosomes, or different transcription regulating regions. For example, a chimeric protein comprising two subsequences, where the subsequences are not associated with each other in nature, or operatively linked to each other in nature, constitutes a protein with mutually heterologous components. A specific example may include, but would not be limited to, a transcriptional activation domain from one protein fused to a transcription factor sequence from another protein, where the two are not associated with each other in nature; in this case, the transcriptional activation domain and the transcription factor sequence are mutually heterologous.

A “polypeptide” is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues for example, at least about 15 consecutive polymerized amino acid residues. In many instances, a polypeptide comprises a polymerized amino acid residue sequence that is a transcription factor or a domain or portion or fragment thereof. Additionally, the polypeptide may comprise: (i) a nuclear localization domain; (ii) an activation domain; (iii) a repression domain; (iv) an oligomerization domain; (v) a protein-protein interaction domain; (vi) a DNA-binding domain; or the like. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.

“Protein” refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.

“Portion”, as used herein, refers to any part of a protein used for any purpose, but especially for the screening of a library of molecules which specifically bind to that portion or for the production of antibodies.

A “recombinant polypeptide” is a polypeptide produced by translation of a recombinant polynucleotide. A “synthetic polypeptide” is a polypeptide created by consecutive polymerization of isolated amino acid residues using methods well known in the art. An “isolated polypeptide,” whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, for example, more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, that is, alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such an enrichment is not the result of a natural response of a wild-type plant. Alternatively, or additionally, the isolated polypeptide is separated from other cellular components with which it is typically associated, for example, by any of the various protein purification methods herein.

“Homology” refers to sequence similarity between a reference sequence and at least a fragment of a newly sequenced clone insert or its encoded amino acid sequence.

“Identity” or “similarity” refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases “percent identity” and “% identity” refer to the percentage of sequence identity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. “Sequence similarity” refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value therebetween. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical, matching or corresponding nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at corresponding positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at corresponding positions shared by the polypeptide sequences.

“Alignment” refers to a number of nucleotide bases or amino acid residue sequences aligned by lengthwise comparison so that components in common (that is, nucleotide bases or amino acid residues at corresponding positions) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences. Alignments such as those of FIG. 1 may be used to identify conserved domains and relatedness within these domains. An alignment may suitably be determined by means of computer programs known in the art, such as MACVECTOR software (1999) (Accelrys, Inc., San Diego, Calif.).

A “conserved domain” or “conserved region” as used herein refers to a region within heterogeneous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity or homology between the distinct sequences. With respect to polynucleotides encoding presently disclosed polypeptides, a conserved domain is preferably at least nine base pairs (bp) in length. Transcription factor sequences that possess or encode for conserved domains that have a minimum percentage identity and have comparable biological activity to the present polypeptide sequences, thus being members of the same clade of transcription factor polypeptides, are encompassed by the invention. Reduced or eliminated expression of a polypeptide that comprises, for example, a conserved domain having DNA-binding, activation or nuclear localization activity, results in the transformed plant having similar improved traits as other transformed plants having reduced or eliminated expression of other members of the same clade of transcription factor polypeptides.

A fragment or domain can be referred to as outside a conserved domain, outside a consensus sequence, or outside a consensus DNA-binding site that is known to exist or that exists for a particular polypeptide class, family, or sub-family. In this case, the fragment or domain will not include the exact amino acids of a consensus sequence or consensus DNA-binding site of a transcription factor class, family or sub-family, or the exact amino acids of a particular transcription factor consensus sequence or consensus DNA-binding site. Furthermore, a particular fragment, region, or domain of a polypeptide, or a polynucleotide encoding a polypeptide, can be “outside a conserved domain” if all the amino acids of the fragment, region, or domain fall outside of a defined conserved domain(s) for a polypeptide or protein. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.

As one of ordinary skill in the art recognizes, conserved domains may be identified as regions or domains of identity to a specific consensus sequence (see, for example, Riechmann et al. (2000) Science 290, 2105-2110; and Riechmann and Ratcliffe (2000) Curr. Opin. Plant Biol. 3, 423-434). Thus, by using alignment methods well known in the art, the conserved domains of the plant polypeptides may be determined.

The conserved domains for many of the polypeptide sequences of the invention are listed in Table 1. Also, the polypeptides of Table 1 have conserved domains specifically indicated by amino acid coordinate start and stop sites. A comparison of the regions of these polypeptides allows one of skill in the art (see, for example, Reeves and Nissen, 1995, to identify domains or conserved domains for any of the polypeptides listed or referred to in this disclosure.

“Complementary” refers to the natural hydrogen bonding by base pairing between purines and pyrimidines. For example, the sequence A-C-G-T (5′->3′) forms hydrogen bonds with its complements A-C-G-T (5′->3′) or A-C-G-U (5′->3′). Two single-stranded molecules may be considered partially complementary, if only some of the nucleotides bond, or “completely complementary” if all of the nucleotides bond. The degree of complementarity between nucleic acid strands affects the efficiency and strength of hybridization and amplification reactions. “Fully complementary” refers to the case where bonding occurs between every base pair and its complement in a pair of sequences, and the two sequences have the same number of nucleotides.

The terms “paralog” and “ortholog” are defined below in the section entitled “Orthologs and Paralogs”. In brief, orthologs and paralogs are evolutionarily related genes that have similar sequences and functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.

The term “equivalog” describes members of a set of homologous proteins that are conserved with respect to function since their last common ancestor. Related proteins are grouped into equivalog families, and otherwise into protein families with other hierarchically defined homology types. This definition is provided at the Institute for Genomic Research (TIGR) World Wide Web (www) website, “tigr.org” under the heading “Terms associated with TIGRFAMs”.

In general, the term “variant” refers to molecules with some differences, generated synthetically or naturally, in their base or amino acid sequences as compared to a reference (native) polynucleotide or polypeptide, respectively. These differences include substitutions, insertions, deletions or any desired combinations of such changes in a native polynucleotide of amino acid sequence.

With regard to polynucleotide variants, differences between presently disclosed polynucleotides and polynucleotide variants are limited so that the nucleotide sequences of the former and the latter are closely similar overall and, in many regions, identical. Due to the degeneracy of the genetic code, differences between the former and latter nucleotide sequences may be silent (that is, the amino acids encoded by the polynucleotide are the same, and the variant polynucleotide sequence encodes the same amino acid sequence as the presently disclosed polynucleotide. Variant nucleotide sequences may encode different amino acid sequences, in which case such nucleotide differences will result in amino acid substitutions, additions, deletions, insertions, truncations or fusions with respect to the similar disclosed polynucleotide sequences. These variations may result in polynucleotide variants encoding polypeptides that share at least one functional characteristic. The degeneracy of the genetic code also dictates that many different variant polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing.

Also within the scope of the invention is a variant of a nucleic acid listed in the Sequence Listing, that is, one having a sequence that differs from the one of the polynucleotide sequences in the Sequence Listing, or a complementary sequence, that encodes a functionally equivalent polypeptide (that is, a polypeptide having some degree of equivalent or similar biological activity) but differs in sequence from the sequence in the Sequence Listing, due to degeneracy in the genetic code. Included within this definition are polymorphisms that may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding polypeptide, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding polypeptide.

“Allelic variant” or “polynucleotide allelic variant” refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations may be “silent” or may encode polypeptides having altered amino acid sequences. “Allelic variant” and “polypeptide allelic variant” may also be used with respect to polypeptides, and in this case, the terms refer to a polypeptide encoded by an allelic variant of a gene.

“Splice variant” or “polynucleotide splice variant” as used herein refers to alternative forms of RNA transcribed from a gene. Splice variation naturally occurs as a result of alternative sites being spliced within a single transcribed RNA molecule or between separately transcribed RNA molecules, and may result in several different forms of mRNA transcribed from the same gene. Thus, splice variants may encode polypeptides having different amino acid sequences, which may or may not have similar functions in the organism. “Splice variant” or “polypeptide splice variant” may also refer to a polypeptide encoded by a splice variant of a transcribed mRNA.

As used herein, “polynucleotide variants” may also refer to polynucleotide sequences that encode paralogs and orthologs of the presently disclosed polypeptide sequences. “Polypeptide variants” may refer to polypeptide sequences that are paralogs and orthologs of the presently disclosed polypeptide sequences.

Differences between presently disclosed polypeptides and polypeptide variants are limited so that the sequences of the former and the latter are closely similar overall and, in many regions, identical. Presently disclosed polypeptide sequences and similar polypeptide variants may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. These differences may produce silent changes and result in functionally equivalent polypeptides. Thus, it will be readily appreciated by those of skill in the art, that any of a variety of polynucleotide sequences is capable of encoding the polypeptides and homolog polypeptides of the invention. A polypeptide sequence variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties. Deliberate amino acid substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as a significant amount of the functional or biological activity of the polypeptide is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine. More rarely, a variant may have “non-conservative” changes, for example, replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing functional or biological activity may be found using computer programs well known in the art, for example, DNASTAR software (see U.S. Pat. No. 5,840,544).

“Fragment”, with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule that retains a usable, functional characteristic. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A “polynucleotide fragment” refers to any subsequence of a polynucleotide, typically, of at least about 9 consecutive nucleotides, preferably at least about 30 nucleotides, more preferably at least about 50 nucleotides, of any of the sequences provided herein. Exemplary polynucleotide fragments are the first sixty consecutive nucleotides of the polynucleotides listed in the Sequence Listing. Exemplary fragments also include fragments that comprise a region that encodes an conserved domain of a polypeptide. Exemplary fragments also include fragments that comprise a conserved domain of a polypeptide.

Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide. Fragments may have uses in that they may have antigenic potential. In some cases, the fragment or domain is a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription. Fragments can vary in size from as few as 3 amino acid residues to the full length of the intact polypeptide, but are preferably at least about 30 amino acid residues in length and more preferably at least about 60 amino acid residues in length.

The invention also encompasses production of DNA sequences that encode polypeptides and derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available nucleic acid constructs and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding polypeptides or any fragment thereof.

The term “plant” includes whole plants, shoot vegetative organs/structures (for example, leaves, stems, rhizomes, and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like), calli, protoplasts, and cells (for example, guard cells, egg cells, and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, multicellular algae, and unicellular algae.

A “control plant” as used in the present invention refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transformed, transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype in the transformed, transgenic or genetically modified plant. A control plant may in some cases be a transformed or transgenic plant line that comprises an empty nucleic acid construct or marker gene, but does not contain the recombinant polynucleotide of the present invention that is expressed in the transformed, transgenic or genetically modified plant being evaluated. In general, a control plant is a plant of the same line or variety as the transformed, transgenic or genetically modified plant being tested. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line used to generate a transformed or transgenic plant herein.

“Wild type” or “wild-type”, as used herein, refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant that has not been genetically modified or treated in an experimental sense. Wild-type cells, seed, components, tissue, organs or whole plants may be used as controls to compare levels of expression and the extent and nature of trait modification with cells, tissue or plants of the same species in which a polypeptide's expression is altered, for example, in that it has been knocked out, overexpressed, or ectopically expressed.

“Transformation” refers to the transfer of a foreign polynucleotide sequence into the genome of a host organism such as that of a plant or plant cell, or introduction of a foreign polynucleotide sequence into plant or plant cell such that is expressed and results in production of protein. Typically, the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et. al. (1987) “Vectors for Cloning in Plant Cells”, Meth. Enzymol., vol. 153:277-292) and biolistic methodology (U.S. Pat. No. 4,945,050 to Klein et al.).

A “transformed plant”, which may also be referred to as a “transgenic plant” or “transformant”, generally refers to a plant, a plant cell, plant tissue, seed or calli that has been through, or is derived from a plant cell that has been through, a stable or transient transformation process in which a “nucleic acid construct” that contains at least one exogenous polynucleotide sequence is introduced into the plant. The “nucleic acid construct” contains genetic material that is not found in a wild-type plant of the same species, variety or cultivar, or may contain extra copies of a native sequence under the control of its native promoter. The genetic material may include a regulatory element, a transgene (for example, a transcription factor sequence), a transgene overexpressing a protein of interest, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, an antisense transgene sequence, a construct containing inverted repeat sequences derived from a gene of interest to induce RNA interference, or a nucleic acid sequence designed to produce a homologous recombination event or DNA-repair based change, or a sequence modified by chimeraplasty. In some embodiments the regulatory and transcription factor sequence may be derived from the host plant, but by their incorporation into a nucleic acid construct, represent an arrangement of the polynucleotide sequences not found in a wild-type plant of the same species, variety or cultivar.

An “untransformed plant” is a plant that has not been through the transformation process.

A “stably transformed” plant, plant cell or plant tissue has generally been selected and regenerated on a selection media following transformation.

A “nucleic acid construct” may comprise a polypeptide-encoding sequence operably linked (that is, under regulatory control of) to appropriate inducible, tissue-specific, developmental, or constitutive regulatory sequences that allow for the controlled expression of polypeptide. The expression vector or cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, for example, a plant explant, to produce a recombinant plant (for example, a recombinant plant cell comprising the nucleic acid construct) as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.

A “trait” refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, for example, by measuring tolerance to water deprivation or particular salt or sugar concentrations, or by the observation of the expression level of a gene or genes, for example, by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as hyperosmotic stress tolerance, disease resistance, growth rate, or yield. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transformed or transgenic plants, however.

“Trait modification” refers to a detectable difference in a characteristic in a plant with reduced or eliminated expression, or ectopic expression, of a polynucleotide or polypeptide of the present invention relative to a plant not doing so, such as a wild-type plant. In some cases, the trait modification can be evaluated quantitatively. For example, the trait modification can entail at least about a 2% increase or decrease, or an even greater difference, in an observed trait as compared with a control or wild-type plant. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution and magnitude of the trait in the plants as compared to control or wild-type plants.

When two or more plants have “similar morphologies”, “substantially similar morphologies”, “a morphology that is substantially similar”, or are “morphologically similar”, the plants have comparable forms or appearances, including analogous features such as overall dimensions, height, width, mass, root mass, shape, glossiness, color, stem diameter, leaf size, leaf dimension, leaf density, internode distance, branching, root branching, number and form of inflorescences, and other macroscopic characteristics, and the individual plants are not readily distinguishable based on morphological characteristics alone.

“Modulates” refers to a change in activity (biological, chemical, or immunological) or lifespan resulting from specific binding between a molecule and either a nucleic acid molecule or a protein.

“Ectopic expression or altered expression” in reference to a polynucleotide indicates that the pattern of expression in, for example, a transformed or transgenic plant or plant tissue, is different from the expression pattern in a wild-type plant or a reference plant of the same species. The pattern of expression may also be compared with a reference expression pattern in a wild-type plant of the same species. For example, the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild-type plant, or by expression at a time other than at the time the sequence is expressed in the wild-type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild-type plant. The term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression. The resulting expression pattern can be transient or stable, constitutive or inducible, tissue specific, or developmentally-regulated (each of these may be controlled by the choice of promoter operably linked to a polynucleotide encoding a polypeptide of the invention). In reference to a polypeptide, the terms “ectopic expression” or “altered expression” further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides.

The term “overexpression” as used herein refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression of that gene in a wild-type plant, cell or tissue, at any developmental or temporal stage. Overexpression can occur when, for example, the genes encoding one or more polypeptides are under the control of a strong promoter (for example, the cauliflower mosaic virus 35S transcription initiation region). Overexpression may also be achieved by placing a gene of interest under the control of an inducible or tissue specific promoter, or may be achieved through integration of transposons or engineered T-DNA molecules into regulatory regions of a target gene. Thus, overexpression may occur throughout a plant, in specific tissues of the plant, or in the presence or absence of particular environmental signals, depending on the promoter or overexpression approach used.

Overexpression may take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to the present polypeptides. Overexpression may also occur in plant cells where endogenous expression of the present polypeptides or functionally equivalent molecules normally occurs, but such normal expression is at a lower level at the same time of day or at the same developmental stage. Overexpression of a gene thus results in a greater than normal production, or “overproduction” of the encoded RNA and or encoded the polypeptide in the plant, cell or tissue.

The term “transcription regulating region” refers to a DNA regulatory sequence that regulates expression of one or more genes in a plant when a transcription factor having one or more specific binding domains binds to the DNA regulatory sequence. Transcription factors typically possess a conserved DNA binding domain. The transcription factors also comprise an amino acid subsequence that forms a transcription activation domain that regulates expression of one or more target genes, such as genes that confer abiotic stress tolerance, in a plant when the transcription factor binds to the regulating region.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The data presented herein represent the results obtained in experiments with polynucleotides and polypeptides that may be expressed in plants for the purpose of increasing yield, or reducing yield losses that arise from abiotic stresses.

The isolation and functional characterization of a small region comprising consecutive amino acids from Ethylene Response Factor 98 (AtERF98, SEQ ID NO: 2) of the flowering plant Arabidopsis is described herein. This small peptide contains many acidic and polar amino acids residue interspersed around hydrophobic leucines. This peptide was designated as the “EDLL motif”, based on four conserved glutamic acid, aspartic acid, leucine and leucine residues in corresponding positions (FIG. 1), and as arranged in the consensus sequence EX₄DX₃LX₃L (SEQ ID NO: 55), where X is any amino acid. The EDLL domain may also be characterized by the consensus sequence E-L/F-X₂₋L/F-D-D/N-X₂-L-X₂-L/M-L (SEQ ID NO: 56), or the consensus sequence E-F/L-X-X-L/F-D-D/N-X-V/L/I-L-X-X-L/M-L (SEQ ID NO: 94), or the consensus sequence E-F/L-E/V-Y/C/F-L/F-D-D/N-X-V/L-L-E/Q/D-E/D/S-L/M-L (SEQ ID NO: 95), where X is any amino acid. Some experimental evidence has been obtained with sequences lacking the glutamic acid residue at the first position, in which the sequences retained some transcriptional activation potential. This modification may represent a means to fine-tune the activation potential of a particular EDLL sequence, which may be useful when a greater or lesser degree of activity of a transcription regulatory polypeptide to which the EDLL domain is fused is desirable.

The EDLL motif is conserved in diverse plant genera including, but not limited to, eudicots including Arabidopsis, Glycine, and Medicago and monocots Oryza, Triticum, and Zea (Table 1 and FIG. 1).

TABLE 1 Gene families and conserved EDLL domains of AtERF98 clade members EDLL SEQ Identical Domains ID NO: residues SEQ GID No./ in AA of EDLL (% ID to G1792 ID NO: Species Coordinates EDLL Domain domain EDLL Domain)  2 AtERF98 117-132 VFEFEYLDDKVLEELL 37 16/16 (100%)  (G1792)/At  4 G1795/At 104-119 VFEFEYLDDSVLEELL 38 15/16 (93.8%)  6 G30/At 100-115 VFEFEYLDDSVLDELL 39 14/16 (87.5%)  8 G1791/At 108-123 VIEFEYLDDSLLEELL 40 13/16 (81.2%) 10 G3520/Gm 109-124 VIEFECLDDKLLEDLL 41 12/16 (75.0%) 12 G3519/Gm 128-143 TFELEYLDNKLLEELL 42 12/16 (75.0%) 14 G3383/Os 101-116 KIEFEYLDDKVLDDLL 43 12/16 (75.0%) 16 G3517/Zm 103-118 VIEFEYLDDEVLQEML 44 12/16 (75.0%) 18 G3518/Gm 135-150 TFELEYFDNKLLEELL 45 11/16 (68.7%) 20 G3739/Zm 107-122 VIELEYLDDEVLQEML 46 11/16 (68.7%) 22 G3736/Ta 108-123 VIEFEYLDDDVLQSML 47 11/16 (68.7%) 24 G3381/Os 109-124 PIEFEYLDDHVLQEML 48 11/16 (68.7%) 26 G3737/Os 101-116 KVELVYLDDKVLDELL 49 11/16 (68.7%) 28 G3515/Os 116-131 KVELECLDDKVLEDLL 50 11/16 (68.7%) 30 G3516/Zm 107-122 KVELECLDDRVLEELL 51 11/16 (68.7%) 32 G3380/Os 103-118 VIELECLDDQVLQEML 52 10/16 (62.5%) 34 G3794/Zm 102-117 VIELECLDDQVLQEML 53 10/16 (62.5%) 36 G3735/Mt 131-144 ELEFLDNKLLQELL 54  9/16 (56.2%) Abbreviations for Table 1: At-Arabidopsis thaliana; Gm-Glycine max; Mt-Medicago truncatula; Os-Oryza sativa; Ta-Triticum aestivum; Zm-Zea mays

By performing a similar analysis starting with each of the EDLL domains in Table 1, the percentage identities of the closest homologs, and the proportion of identical residues (in parentheses), in decreasing order of identity to the following EDLL domains, are, for the:

G1795 EDLL domain, SEQ ID NO: 38, the following share identical residues of:

93.8% (15/16)—AtERF98, G30;

87.5% (14/16)—G1791;

75.0% (12/16)—G3517;

68.7% (11/16)—G3736, G3383, G3381, G3739, G3519, G3520, G3516;

62.5% (10/16)—G3518, G3794, G3737, G3380, G3515; and

50.0% (8/16)—G3735;

G30 EDLL domain, SEQ ID NO: 39, the following share identical residues of:

93.8% (15/16)—G1795;

87.5% (14/16)—G1792;

81.2% (13/16)—G1791;

75.0% (12/16)—G3383, G3517;

68.7% (11/16)—G3736, G3381, G3739, G3737;

62.5% (10/16)—G3519, G3520, G3794, G3380, G3516;

56.2% (9/16)—G3518, G3515; and

50.0% (8/16)—G3735;

G1791 EDLL domain, SEQ ID NO: 40, the following share identical residues of:

87.5% (14/16)—G1795;

81.2% (13/16)—G30, AtERF98, G3520;

75.0% (12/16)—G3517;

68.7% (11/16)—G3736, G3383, G3381, G3739, G3519;

62.5% (10/16)—G3794, G3518, G3380, G3516;

56.2% (9/16)—G3737, G3735, G3515; and

50.0% —(8/16);

G3520 EDLL domain, SEQ ID NO: 41, the following share identical residues of:

81.2% (13/16)—G1791;

75.0% (12/16)—AtERF98, G3515, G3383;

68.7% (11/16)—G1795;

62.5% (10/16)—G30, G3516, G3794, G3380, G3517, G3736, G3519; and

56.2% (9/16)—G3739, G3381, G3735, G3518, G3737;

G3519 EDLL domain, SEQ ID NO: 42, the following share identical residues of:

93.8% (15/16)—G3518;

75.0% (12/16)—AtERF98, G3735;

68.7% (11/16)—G1795, G1791;

62.5% (10/16)—G30, G3737, G3516, G3515, G3520;

56.2% (9/16)—G3739, G3383;

50.0% (8/16)—G3517, G3381, G3794, G3380; and

43.7% (7/16)—G3736;

G3383 EDLL domain, SEQ ID NO: 43, the following share identical residues of:

75.0% (12/16)—AtERF98, G30, G3737, G3515, G3520;

68.7% (11/16)—G1791, G1795, G3381, G3517, G3736;

62.5% (10/16)—G3516, G3739;

56.2% (9/16)—G3380, G3794, G3519; and

50.0% (8/16)—G3518, G3735;

G3517 EDLL domain, SEQ ID NO: 44, the following share identical residues of:

93.8% (15/16)—G3739;

87.5% (14/16)—G3736, G3381;

81.2% (13/16)—G3380, G3794;

75.0% (12/16)—AtERF98, G30, G1791, G1795;

68.7% (11/16)—G3383;

62.5% (10/16)—G3520;

56.2% (9/16)—G3737, G3516;

50.0% (8/16)—G3735, G3515, G3519; and

43.7% (7/16)—G3518;

G3517 EDLL domain, SEQ ID NO: 45, the following share identical residues of:

93.8% (15/16)—G3519;

68.7% (11/16)—AtERF98, G3735;

62.5% (10/16)—G1791, G1795;

56.2% (9/16)—G30, G3515, G3516, G3520, G3737;

50.0% (8/16)—G3383, G3739;

43.7% (7/16)—G3380, G3381, G3517, G3794; and

37.5% (6/16)—G3736;

G3739 EDLL domain, SEQ ID NO: 46, the following share identical residues of:

93.8% (15/16)—G3517;

87.5% (14/16)—G3380, G3794;

81.2% (13/16)—G3381, G3736;

68.7% (11/16)—AtERF98, G30, G1791, G1795;

62.5% (10/16)—G3383, G3737, G3516;

56.2% (9/16)—G3515, G3519, G3520, G3735; and

50.0% (8/16)—G3518;

G3739 EDLL domain, SEQ ID NO: 47, the following share identical residues of:

87.5% (14/16)—G3517;

81.2% (13/16)—G3381, G3739;

75.0% (12/16)—G3380, G3794;

68.7% (11/16)—AtERF98, G30, G1791, G1795, G3383;

62.5% (10/16)—G3520;

50.0% (8/16)—G3515, G3516; G3737;

43.7% (7/16)—G3519, G3735;

37.5% (6/16)—G3518;

G3381 EDLL domain, SEQ ID NO: 48, the following share identical residues of:

87.5% (14/16)—G3517;

81.2% (13/16)—G3736, G3739;

75.0% (12/16)—G3380, G3794;

68.7% (11/16)—AtERF98, G30, G1791, G1795, G3383;

56.2% (9/16)—G3516, G3520, G3737;

50.0% (8/16)—G3515, G3519, G3735; and

43.7% (7/16)—G3518;

G3737 EDLL domain, SEQ ID NO: 49, the following share identical residues of:

75.0% (12/16)—G3383, G3515, G3516;

68.7% (11/16)—AtERF98, G30;

62.5% (10/16)—G1795, G3519, G3739;

56.2% (9/16)—G1791, G3380, G3381, G3517, G3518, G3735, G3794; and

50.0% (8/16)—G3520, G3736;

G3515 EDLL domain, SEQ ID NO: 50, the following share identical residues of:

87.5% (14/16)—G3516;

75.0% (12/16)—G3383, G3520, G3737;

68.7% (11/16)—AtERF98;

62.5% (10/16)—G1795, G3380, G3519, G3794;

56.2% (9/16)—G30, G1791, G3518, G3735, G3739; and

50.0% (8/16)—G3381, G3517, G3736;

G3516 EDLL domain, SEQ ID NO: 51, the following share identical residues of:

87.5% (14/16)—G3515;

75.0% (12/16)—G3737;

68.7% (11/16)—AtERF98, G1795, G3380, G3794;

62.5% (10/16)—G30, G1791, G3383, G3519, G3520, G3739;

56.2% (9/16)—G3381, G3517, G3518, G3735; and

50.0% (8/16)—G3736;

G3380 EDLL domain, SEQ ID NO: 52, the following share identical residues of:

100% (16/16)—G3794;

87.5% (14/16)—G3739;

81.2% (13/16)—G3517;

75.0% (12/16)—G3381, G3736;

68.7% (11/16)—G3516;

62.5% (10/16)—AtERF98, G30, G1791, G1795, G3515, G3520;

56.2% (9/16)—G3383, G3735, G3737;

50.0% (8/16)—G3519; and

43.7% (7/16)—G3518;

G3794 EDLL domain, SEQ ID NO: 53, the following share identical residues of:

100% (16/16)—G3380;

87.5% (14/16)—G3739;

81.2% (13/16)—G3517;

75.0% (12/16)—G3381, G3736;

68.7% (11/16)—G3516;

62.5% (10/16)—AtERF98, G30, G1791, G1795, G3515, G3520;

56.2% (9/16)—G3383, G3735, G3737;

50.0% (8/16)—G3519; and

43.7% (7/16)—G3518;

G3735 EDLL domain, SEQ ID NO: 54, the following share identical residues of:

75.0% (12/16)—G3519;

68.7% (11/16)—G3518;

56.2% (9/16)—AtERF98, G1791, G3380, G3515, G3516, G3520, G3737, G3739, G3794;

50.0% (8/16)—G30, G1795, G3381, G3383, G3517;

43.7% (7/16)—G3736.

Since the EDLL motif has many acidic residues, it was predicted by us to have role in transcriptional activation. The present application confirms the transcriptional activation potential and transportability of function of this small peptide experimentally. To demonstrate experimentally the role of conserved EDLL motif, we fused a 24 amino acid peptide sequence comprising the EDLL domain of AtERF98 with a sequence-specific GAL4 DNA binding domain (DBD) from yeast (GAL4 DBD or “GD”). The chimeric protein, (GD-EDLL) when expressed in plant protoplasts, induced the expression of a reporter gene containing GAL4 DBD binding sequences in the promoter (FIG. 2). The GAL4 DBD alone without the EDLL motif (GD) could not induce the expression of the reporter gene significantly (FIG. 2). The activation of the reporter gene by the either one or two copies of the EDLL motif is comparable in magnitude to that obtained with the widely used VP16 activation domain from herpes simplex virus (FIGS. 2, 3). When the hydrophobic leucine residues were changed to valine (“EDLLm”), the activation potential of EDLL motif was significantly compromised (FIG. 2). Similarly, when orthologous EDLL motifs from Medicago truncatula (GD:G3735EDLL) and rice (GD:G3737EDLL) were tested, each produced reporter gene levels significantly higher than the GALA DNA binding domain alone (GD) (FIG. 3). Other orthologous EDLL motif sequences from crops such as corn, soybean and wheat will be tested in a similar manner.

We have also shown another example (FIG. 4) where an EDLL domain was fused to NF-YB1 (G481, SEQ ID NO: 73), a protein which lacks a native strong activation domain of its own and which does not bind DNA alone, but rather requires a DNA binding partner for recruitment to the promoter. When the G481:EDLL fusion was co-expressed in protoplasts with a yeast GALA DNA binding domain (GD) fused to G483 (SEQ ID NO: 74, the G481:EDLL-GD:G483 dimer induced the activity of reporter gene to a significant degree. Similar results were obtained with another NF-YC protein, G715, SEQ ID NO: 75, and the result was similar to that with G483. These results demonstrated the utility of the EDLL domain in activating transcription by way of a CCAAT element binding factor, which comprises a transcription regulatory polypeptide unrelated to the sequence from which the EDLL domain was derived (AtERF98). This also indicated that the EDLL motif can function in larger complexes, and can confer transcriptional activation function to a plant transcription factor lacking activation capacity. It is also active even if the protein is not binding DNA directly (G481:EDLL alone can not bind DNA; data not shown) but is recruited to the DNA via interaction with another DNA binding protein (GD:G483 or GD:715).

Orthologs and Paralogs

Homologous sequences as described above, such as sequences that are homologous to AtERF98 (SEQ ID NO: 2), or the EDLL domain of AtERF98 (SEQ ID NO: 37), can include orthologous or paralogous sequences (for example, SEQ ID NOs: 1-36, or EDLL domains 37-54). Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. General methods for identifying orthologs and paralogs, including phylogenetic methods, sequence similarity and hybridization methods, are described herein; an ortholog or paralog, including equivalogs, may be identified by one or more of the methods described below.

As described by Eisen (1998) Genome Res. 8: 163-167, evolutionary information may be used to predict gene function. It is common for groups of genes that are homologous in sequence to have diverse, although usually related, functions. However, in many cases, the identification of homologs is not sufficient to make specific predictions because not all homologs have the same function. Thus, an initial analysis of functional relatedness based on sequence similarity alone may not provide one with a means to determine where similarity ends and functional relatedness begins. Fortunately, it is well known in the art that protein function can be classified using phylogenetic analysis of gene trees combined with the corresponding species. Functional predictions can be greatly improved by focusing on how the genes became similar in sequence (that is, by evolutionary processes) rather than on the sequence similarity itself (Eisen, supra). In fact, many specific examples exist in which gene function has been shown to correlate well with gene phylogeny (Eisen, supra). Thus, “[t]he first step in making functional predictions is the generation of a phylogenetic tree representing the evolutionary history of the gene of interest and its homologs. Such trees are distinct from clusters and other means of characterizing sequence similarity because they are inferred by techniques that help convert patterns of similarity into evolutionary relationships . . . . After the gene tree is inferred, biologically determined functions of the various homologs are overlaid onto the tree. Finally, the structure of the tree and the relative phylogenetic positions of genes of different functions are used to trace the history of functional changes, which is then used to predict functions of [as yet] uncharacterized genes” (Eisen, supra).

Within a single plant species, gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs. A paralog is therefore a similar gene formed by duplication within the same species. Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) Methods Enzymol. 266: 383-402). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle (1987) J. Mol. Evol. 25: 351-360). For example, a clade of very similar MADS domain transcription factors from Arabidopsis all share a common function in flowering time (Ratcliffe et al. (2001) Plant Physiol. 126: 122-132, and a group of very similar AP2 domain transcription factors from Arabidopsis are involved in tolerance of plants to freezing (Gilmour et al. (1998) Plant J. 16: 433-442). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the clade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function (see also, for example, Mount (2001), in Bioinformatics: Sequence and Genome Analysis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., p. 543)

Transcription factor gene sequences are conserved across diverse eukaryotic species lines (Goodrich et al. (1993) Cell 75: 519-530; Lin et al. (1991) Nature 353: 569-571; Sadowski et al. (1988) Nature 335: 563-564). Plants are no exception to this observation; diverse plant species possess transcription factors that have similar sequences and functions. Speciation, the production of new species from a parental species, gives rise to two or more genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a corresponding orthologous gene in another plant species. Once a phylogenic tree for a gene family of one species has been constructed using a program such as CLUSTAL (Thompson et al., 1994, supra; Higgins et al., 1996, supra) potential orthologous sequences can be placed into the phylogenetic tree and their relationship to genes from the species of interest can be determined. Orthologous sequences can also be identified by a reciprocal BLAST strategy. Once an orthologous sequence has been identified, the function of the ortholog can be deduced from the identified function of the reference sequence.

Thus, the invention provides methods for identifying a sequence similar or paralogous or orthologous or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an internet or intranet) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.

In addition, one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to search against a BLOCKS (Bairoch et al. (1997) Nucleic Acids Res. 25: 217-221), PFAM, and other databases which contain previously identified and annotated motifs, sequences and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al. (1992) Protein Engineering 5: 35-51) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul (1990) J. Mol. Biol. 215: 403-410, and Altschul (1993) J. Mol. Evol. 36: 290-300), BLOCKS (Henikoff and Henikoff (1991) Nucleic Acids Res. 19: 6565-6572), Hidden Markov Models (HMM; Eddy (1996) Curr. Opin. Str. Biol. 6: 361-365; Sonnhammer et al. (1997) Proteins 28: 405-420), and the like, can be used to manipulate and analyze polynucleotide and polypeptide sequences encoded by polynucleotides. These databases, algorithms and other methods are well known in the art and are described in Ausubel et al. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., unit 7.7, and in Meyers (1995) Molecular Biology and Biotechnology, Wiley VCH, New York, N.Y., p 856-853.

Methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and conserved domains characteristic of a particular transcription factor family. Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiary structure between a polypeptide sequence encoded by a polynucleotide that comprises a known function and a polypeptide sequence encoded by a polynucleotide sequence that has a function not yet determined. Such examples of tertiary structure may comprise predicted alpha helices, beta-sheets, amphipathic helices, leucine zipper motifs, zinc finger motifs, proline-rich regions, cysteine repeat motifs, and the like.

EDLL domains of presently disclosed polypeptides may be cloned using compositions provided by the present invention according to methods well known in the art. cDNAs can be cloned using mRNA from a plant cell or tissue that expresses one of the present sequences. Appropriate mRNA sources may be identified by interrogating Northern blots with probes designed from the present sequences, after which a library is prepared from the mRNA obtained from a positive cell or tissue. Polypeptide-encoding cDNA is then isolated using, for example, PCR, using primers designed from a presently disclosed gene sequence, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA library may be used to transform plant cells. Expression of the cDNAs of interest is detected using, for example, microarrays, Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may be isolated using similar techniques to those.

Examples of EDLL domains from polypeptide sequences of Arabidopsis and other plant species are listed in Table 1 and in the Sequence Listing as SEQ ID NOs: 37-54. In addition to the sequences in Table 1 and the Sequence Listing, the invention includes, but is not limited to, isolated polypeptide sequences that are phylogenetically and structurally similar to EDLL sequences listed in Table 1, and in the Sequence Listing as SEQ ID NOs: 37-54, and can function in a plant as a transcriptional activation domain, or by activating gene transcription and increasing the expression of a protein in a living organism or in vitro gene or protein expression system. The invention includes, but is not limited to, protein sequences that are found in the Sequence Listing as SEQ ID NOs: 2n, where n=1-18, or structurally similar sequences, when the sequences include an EDLL domain that functions as a transcriptional activation domain.

Sequence Variations

It will readily be appreciated by those of skill in the art, that any of a variety of polynucleotide sequences are capable of encoding the transcription factors and transcription factor homolog polypeptides that function similarly to those provided in the Sequence Listing or Table 1. Due to the degeneracy of the genetic code, many different polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing. Nucleic acids having a sequence that differs from the sequences shown in the Sequence Listing, or complementary sequences, that encode functionally equivalent peptides (that is, peptides having some degree of equivalent or similar biological activity) but differ in sequence from the sequence shown in the sequence listing due to degeneracy in the genetic code, are also within the scope of the invention.

Altered polynucleotide sequences encoding polypeptides include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide encoding a polypeptide with at least one functional characteristic of the instant polypeptides. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding the instant polypeptides, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding the instant polypeptides.

Sequence alterations that do not change the amino acid sequence encoded by the polynucleotide are termed “silent” variations. With the exception of the codons ATG and TGG, encoding methionine and tryptophan, respectively, any of the possible codons for the same amino acid can be substituted by a variety of techniques, for example, site-directed mutagenesis, available in the art. Accordingly, any and all such variations of a sequence selected from the above table are a feature of the invention.

In addition to silent variations, other conservative variations that alter one, or a few amino acids in the encoded polypeptide, can be made without altering the function of the polypeptide. For example, substitutions, deletions and insertions introduced into the sequences provided in the Sequence Listing are also envisioned. Such sequence modifications can be engineered into a sequence by site-directed mutagenesis (for example, Olson et al., Smith et al., Zhao et al., and other articles in Wu (ed.) Meth. Enzymol. (1993) vol. 217, Academic Press) or the other methods known in the art or noted herein. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In preferred embodiments, deletions or insertions are made in adjacent pairs, for example, a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a sequence. The mutations that are made in the polynucleotide encoding the transcription factor should not place the sequence out of reading frame and should not create complementary regions that could produce secondary mRNA structure. Preferably, the polypeptide encoded by the DNA performs the desired function.

Conservative substitutions are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 2 when it is desired to maintain the activity of the protein. Table 2 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as conservative substitutions.

TABLE 2 Possible conservative amino acid substitutions Amino Acid Conservative Residue substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Gln Asn Cys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr Tyr Trp; Phe Val Ile; Leu

The EDLL domains provided in the Sequence Listing or in Table 1 have a novel activity, being plant transcription activation domains that may be used to activate transcription of heterologous transcription regulatory proteins. Although all conservative amino acid substitutions (for example, one basic amino acid substituted for another basic amino acid) in the EDLL domain will not necessarily result in a protein that has transcriptional activation activity, it is expected that many of these conservative mutations would result in an EDLL domain having transcriptional activation activity. Most mutations, conservative or non-conservative, made to a protein having an EDLL domain, but outside of the EDLL domain and outside of other domains essential for protein activity, will not affect the activity of the EDLL domain to any great extent.

Identifying Polynucleotides or Polypeptides Related to the Disclosed Sequences by Percent Identity

With the aid of a computer, one of skill in the art could identify all of the polypeptides, or all of the nucleic acids that encode a polypeptide, with, for example, at least 85% identity to the sequences provided herein and in the Sequence Listing. Electronic analysis of sequences may be conducted with a software program such as the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, for example, the clustal method (see, for example, Higgins and Sharp (1988) Gene 73: 237-244). The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. Other alignment algorithms or programs may be used, including FASTA, BLAST, or ENTREZ, FASTA and BLAST, and which may be used to calculate percent similarity. These are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with or without default settings. ENTREZ is available through the National Center for Biotechnology Information. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, for example, each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences (see U.S. Pat. No. 6,262,333).

Software for performing BLAST analyses is publicly available, for example, through the National Center for Biotechnology Information (see internet website at www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, 1990, supra; Altschul et al., 1993, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915). Unless otherwise indicated for comparisons of predicted polynucleotides, “sequence identity” refers to the % sequence identity generated from a tblastx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter “off” (see, for example, internet website at www.ncbi.nlm.nih.gov/).

Other techniques for alignment are described by Doolittle, ed. (1996) Methods in Enzymology, vol. 266: “Computer Methods for Macromolecular Sequence Analysis” Academic Press, Inc., San Diego, Calif., USA. Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments (see Shpaer (1997) Methods Mol. Biol. 70: 173-187). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.

Percent identity can also be determined manually, by comparing the entire length of a sequence of sequence with another in an optimal alignment.

Generally, the percentage similarity between two polypeptide sequences, for example, sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between polynucleotide sequences can also be counted or calculated by other methods known in the art, for example, the Jotun Hein method (see, for example, Hein (1990) Methods Enzymol. 183: 626-645). Identity between sequences can also be determined by other methods known in the art, for example, by varying hybridization conditions (see US Patent Application No. US20010010913).

At the polynucleotide level, the sequences described herein in the Sequence Listing, and the sequences of the invention by virtue of a paralogous or homologous relationship with the sequences described in the Sequence Listing, will typically share at least about 30%, or 40% nucleotide sequence identity, preferably at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to one or more of the listed full-length sequences, or to a region of a listed sequence excluding or outside of the region(s) encoding a known consensus sequence or consensus DNA-binding site, or outside of the region(s) encoding one or all conserved domains. The degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein.

At the polypeptide level, the sequences described herein in the Sequence Listing and Table 1, and the sequences of the invention by virtue of a paralogous or homologous relationship with the sequences described in the Sequence Listing or in Table 1, will typically share at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% amino acid sequence identity or more sequence identity to one or more of the listed full-length sequences, including full-length and EDLL domain sequences, or to a listed sequence but excluding or outside of the known consensus sequence or consensus DNA-binding site.

Identifying Polynucleotides Related to the Disclosed Sequences by Hybridization

Polynucleotides homologous to the sequences illustrated in the Sequence Listing and tables can be identified, for example, by hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations (and number thereof), as described in more detail in the references cited below (for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Schroeder et al. (2002) Current Biol. 12, 1462-1472; Berger and Kimmel (1987), “Guide to Molecular Cloning Techniques”, in Methods in Enzymology, vol. 152, Academic Press, Inc., San Diego, Calif.; and Anderson and Young (1985) “Quantitative Filter Hybridisation”, In: Hames and Higgins, ed., Nucleic Acid Hybridisation, A Practical Approach. Oxford, IRL Press, 73-111).

Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger (1987) Methods Enzymol. 152: 399-407; and Kimmel (1987) Methods Enzymol. 152: 507-511). In addition to the nucleotide sequences listed in the Sequence Listing, full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods. The cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.

With regard to hybridization, conditions that are highly stringent, and means for achieving them, are well known in the art. See, for example, Sambrook et al., 1989; Berger, 1987, pages 467-469; and Anderson and Young, 1985, all supra.

Stability of DNA duplexes is affected by such factors as base composition, length, and degree of base pair mismatch. Hybridization conditions may be adjusted to allow DNAs of different sequence relatedness to hybridize. The melting temperature (T_(m)) is defined as the temperature when 50% of the duplex molecules have dissociated into their constituent single strands. The melting temperature of a perfectly matched duplex, where the hybridization buffer contains formamide as a denaturing agent, may be estimated by the following equations:

T _(m)(° C.)=81.5+16.6(log [Na+])+0.41(% G+C)−0.62(% formamide)−500/L  (I) DNA-DNA:

T _(m)(° C.)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(% G+C)²−0.5(% formamide)−820/L  (II) DNA-RNA:

T _(m)(° C.)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(% G+C)²−0.35(% formamide)−820/L  (III) RNA-RNA:

where L is the length of the duplex formed, [Na+] is the molar concentration of the sodium ion in the hybridization or washing solution, and % G+C is the percentage of (guanine+cytosine) bases in the hybrid. For imperfectly matched hybrids, approximately 1° C. is required to reduce the melting temperature for each 1% mismatch.

Hybridization experiments are generally conducted in a buffer of pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson and Young, 1985, supra). In addition, one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non-complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS), polyvinyl-pyrrolidone, ficoll and Denhardt's solution. Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time. In some instances, conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.

Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments such as genes that duplicate functional enzymes from closely related organisms. The stringency can be adjusted either during the hybridization step or in the post-hybridization washes. Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency (as described by the formula above). As a general guidelines high stringency is typically performed at T_(m)−5° C. to T_(m)20° C., moderate stringency at T_(m)−20° C. to T_(m)−35° C. and low stringency at T_(m)−35° C. to T_(m)50° C. for duplex >150 base pairs. Hybridization may be performed at low to moderate stringency (25-50° C. below T_(m)), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at T_(m)25° C. for DNA-DNA duplex and T_(m)−15° C. for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.

High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences. An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or Northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5° C. to 20° C. lower than the thermal melting point (T_(m)) for the specific sequence at a defined ionic strength and pH. Conditions used for hybridization may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about 0.1% N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at hybridization temperatures between about 50° C. and about 70° C. More preferably, high stringency conditions are about 0.02 M sodium chloride, about 0.5% casein, about 0.02% SDS, about 0.001 M sodium citrate, at a temperature of about 50° C. Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire DNA molecule or selected portions, for example, to a unique subsequence, of the DNA.

Stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate. Increasingly stringent conditions may be obtained with less than about 500 mM NaCl and 50 mM trisodium citrate, to even greater stringency with less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, for example, formamide, whereas high stringency hybridization may be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. with formamide present. Varying additional parameters, such as hybridization time, the concentration of detergent, for example, sodium dodecyl sulfate (SDS) and ionic strength, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed.

The washing steps that follow hybridization may also vary in stringency; the post-hybridization wash steps primarily determine hybridization specificity, with the most critical factors being temperature and the ionic strength of the final wash solution. Wash stringency can be increased by decreasing salt concentration or by increasing temperature. Stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.

Thus, hybridization and wash conditions that may be used to bind and remove polynucleotides with less than the desired homology to the nucleic acid sequences or their complements that encode the present polypeptides include, for example:

6×SSC and 1% SDS at 65° C.;

50% formamide, 4×SSC at 42° C.; or

0.5×SSC to 2.0×SSC, 0.1% SDS at 50° C. to 65° C.;

with a first wash step of, for example, 10 minutes at about 42° C. with about 20% (v/v) formamide in 0.1×SSC, and with, for example, a subsequent wash step with 0.2×SSC and 0.1% SDS at 65° C. for 10, 20 or 30 minutes. An example of an amino acid sequence of the invention would include one encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 57-63 (nucleic acid sequence fragments encoding various EDLL domain that have been or can be used for cloning) and 76-93 (nucleic acid sequence fragments that encode various EDLL domains, and which can be incorporated into nucleic acid constructs for cloning purposes).

Useful variations on these conditions will be readily apparent to those skilled in the art.

A person of skill in the art would not expect substantial variation among polynucleotide species encompassed within the scope of the present invention because the highly stringent conditions set forth in the above formulae yield structurally similar polynucleotides.

If desired, one may employ wash steps of even greater stringency, including about 0.2×SSC, 0.1% SDS at 65° C. and washing twice, each wash step being about 30 minutes, or about 0.1×SSC, 0.1% SDS at 65° C. and washing twice for 30 minutes. The temperature for the wash solutions will ordinarily be at least about 25° C., and for greater stringency at least about 42° C. Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3° C. to about 5° C., and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6° C. to about 9° C. For identification of less closely related homologs, wash steps may be performed at a lower temperature, for example, 50° C.

An example of a low stringency wash step employs a solution and conditions of at least 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS over 30 minutes. Greater stringency may be obtained at 42° C. in 15 mM NaCl, with 1.5 mM trisodium citrate, and 0.1% SDS over 30 minutes. Even higher stringency wash conditions are obtained at 65° C.-68° C. in a solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Wash procedures will generally employ at least two final wash steps. Additional variations on these conditions will be readily apparent to those skilled in the art (see, for example, US Patent Application No. US20010010913).

Stringency conditions can be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-10× higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid encoding a polypeptide known as of the filing date of the application. It may be desirable to select conditions for a particular assay such that a higher signal to noise ratio, that is, about 15× or more, is obtained. Accordingly, a subject nucleic acid will hybridize to a unique coding oligonucleotide with at least a 2× or greater signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide. The particular signal will depend on the label used in the relevant assay, for example, a fluorescent label, a colorimetric label, a radioactive label, or the like. Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger, 1987, pages 399-407; and Kimmel, 1987). In addition to the nucleotide sequences in the Sequence Listing, full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods. The cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.

Examples

It is to be understood that this invention is not limited to the particular devices, machines, materials and methods described. Although particular embodiments are described, equivalent embodiments may be used to practice the invention.

The invention, now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention. It will be recognized by one of skill in the art that a polypeptide that is associated with a particular first trait may also be associated with at least one other, unrelated and inherent second trait which was not predicted by the first trait.

Example I. Identification of the EDLL Domain in Diverse Plant Species

Initial examination of the AtERF98 transcription factor sequence revealed the presence of a putative activation domain based on the presence of a high proportion of acidic and polar amino acids residue interspersed around hydrophobic leucines in a short stretch of the sequence near its c-terminus.

Of particular interest to us was whether this domain might exist, and function in a similar manner, in the form of homologs in plant species other than Arabidopsis. Homologous putative activation domains from Arabidopsis and other plant species were next identified using database sequence search tools, such as the Basic Local Alignment Search Tool (BLAST) (Altschul et al. (1990) supra; and Altschul et al. (1997) Nucleic Acid Res. 25: 3389-3402). tblastx sequence analysis programs were employed using the BLOSUM-62 scoring matrix (Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919). The NCBI GenBank database was filtered for sequences by selecting all entries in the NCBI GenBank database associated with NCBI taxonomic ID 33090 (Viridiplantae; all plants). These sequences were compared to the AtERF98 EDLL domain sequence using the Washington University TBLASTX algorithm at the default settings using gapped alignments with the filter “off”. Individual comparisons were ordered by probability score (P-value), where the score reflected the probability that a particular alignment occurred by chance. In addition to P-values, comparisons were also scored by percentage identity. Percentage identity reflects the degree to which two segments of DNA or protein are identical over a particular length. Paralogous or orthologous EDLL domain sequences were readily identified. Examples of sequences so identified are presented in Table 1.

Candidate paralogous and orthologous sequences were identified from proprietary unigene sets of plant gene sequences in Zea mays, Glycine max, Oryza sativa, Triticum aestivum, and Medicago truncatula based on significant homology to the AtERF98 EDLL domain sequence. These candidate EDLL motifs were reciprocally compared to the AtERF98 EDLL domain using a similar BLAST analysis. If the candidate EDLL domain showed maximal similarity to the eliciting EDLL domain, then it was considered to be an ortholog or paralog. Identified Arabidopsis and non-Arabidopsis sequences that were shown in this manner to be orthologous to the Arabidopsis sequences are provided in Table 1.

It is expected that the same methods may be applied to identify other useful and valuable EDLL domain sequences, and the EDLL domain sequences may be derived from a diverse range of species.

The percent sequence identity among the identified EDLL domain sequences examined thus far can be as low as 37.5% (6 of 16 residues identical), as indicated in Table 1 and the subsequent text provided above. Each of these sequences was discovered to have several highly conserved residues, as shown in FIG. 1. These include, in order from N- to C termini, the four residues of glutamic acid, aspartic acid, leucine and leucine residues as indicated in the consensus sequence SEQ ID NO: 55: EX₄DX₃LX₃L, where X can be any amino acid. This peptide was thus designated as the “EDLL domain” (sometimes referred to as the “EDLL motif”), based on these four conserved residues. In addition to the glutamic acid, aspartic acid, leucine and leucine residues, several other positions in this domain were recognized as highly conserved, exemplified by the consensus sequence E-L/F-X₂₋L/F-D-D/N-X₂-L-X₂-L/M-L (SEQ ID NO: 56), or the consensus sequence E-F/L-X-X-L/F-D-D/N-X-V/L/I-L-X-X-L/M-L (SEQ ID NO: 94), or the consensus sequence E-F/L-E/V-Y/C/F-L/F-D-D/N-X-V/L-L-E/Q/D-E/D/S-L/M-L (SEQ ID NO: 95), where X is any amino acid and a “slash” indicates the possibility of alternative residues on either side of the slash (or slashes) at a given position. For example, L/F refers to a leucine or phenylalanine residue, D/N refers to a aspartic acid or asparagine residue, L/M refers to a leucine or methionine residue, and C/F/Y refers to a cysteine residue, a phenylalanine residue, or a tyrosine residue at the indicated position.

Example II. Transformation Methods

Transformation of Arabidopsis with a nucleic acid constructs, such as a construct encoding an EDLL domain, is performed by an Agrobacterium-mediated protocol based on the method of Bechtold and Pelletier (1998) Methods Mol. Biol. 82: 259-266. Unless otherwise specified, all experimental work is done using the Columbia ecotype.

Plant Preparation.

Arabidopsis seeds are sown on mesh covered pots. The seedlings are thinned so that 6-10 evenly spaced plants remain on each pot 10 days after planting. The primary bolts are cut off a week before transformation to break apical dominance and encourage auxiliary shoots to form. Transformation is typically performed at 4-5 weeks after sowing.

Bacterial Culture Preparation.

Agrobacterium stocks are inoculated from single colony plates or from glycerol stocks and grown with the appropriate antibiotics and grown until saturation. On the morning of transformation, the saturated cultures are centrifuged and bacterial pellets are re-suspended in Infiltration Media (0.5×MS, 1×B5 Vitamins, 5% sucrose, 1 mg/ml benzylaminopurine riboside, 200 μl/L Silwet L77) until an A600 reading of 0.8 is reached.

Transformation and Seed Harvest.

The Agrobacterium solution is poured into dipping containers. All flower buds and rosette leaves of the plants are immersed in this solution for 30 seconds. The plants are laid on their side and wrapped to keep the humidity high. The plants are kept this way overnight at 22° C. and then the pots are turned upright, unwrapped, and moved to the growth racks.

The plants are maintained on a growth rack under 24-hour light until seeds are ready to be harvested. Seeds are harvested when 80% of the siliques of the transformed plants are ripe (approximately 5 weeks after the initial transformation). This transformed seed is deemed T0 seed, since it is obtained from the T0 generation, and is later plated on selection plates (typically either kanamycin or sulfonamide, depending on the selectable marker gene included in the transformation construct). Resistant plants that are identified on such selection plates comprise the T1 generation.

Example III. Protoplast-Based Transcriptional Activation Assays

Carrot (Daucus carota) protoplasts were isolated from suspension cultures and transfected essentially by the method of Liu (1994) Plant Cell 6: 645-657. Briefly, plant protoplasts were prepared from a carrot suspension culture maintained at log phase in “carrot suspension medium” (CSM). A fresh culture was prepared by inoculating 50 ml fresh CSM media with 5 mL of 7-day old suspension cell culture and grown 5 days at room temperature. The suspension cells were collected by centrifugation (1000 rpm, 3 min) and resuspended in an equal volume of Driselase solution (Sigma-Aldrich). Driselase, a mixture of fungal enzymes, hydrolyzes cellulose (to glucose) and all the major matrix polysaccharides (to monosaccharides and/or characteristic disaccharides). The suspension culture was poured into 15 mm Petri dishes and incubated 3 h at room temperature. The protoplasts were filtered through a nylon membrane and washed twice with a W5 solution. Each time the protoplasts were pelleted by centrifugation (100 rpm, 3 min) and resuspended by gentle inversion. The final solution was then incubated on ice for 30 min. Prior to transformation, the protoplast cells were pelleted and resuspended in MC solution to a final concentration of 2×10⁶ cells/ml, usually 25-30 ml. Approximately 5×10⁵ cells (300 μl of the suspension) were transformed by adding 10 μg of high quality plasmid DNA and an equal volume of 40% PEG, swirled gently and incubated at room temperature for 20 min. The solution was then diluted to 5 ml using CSM media and incubated an additional 16-18 h to allow for protein expression. The protoplasts were pelleted by centrifugation (1000 rpm, 3 min), the cells disrupted in lysis buffer and the sample assayed for GUS activity by the method of Liu et al, 1995, supra. At least three replicate transfections were performed for each set of constructs analyzed.

Sequences to be analyzed for transcriptional activation potential were fused to a sequence-specific GAL4 DNA binding domain (GAL4DBD or GD in the text) from yeast. The GAL4 DNA binding domain lacks any activation sequence; hence alone it can not activate the transcription of any gene. This construct was co-transfected with a reporter construct containing GAL4 binding sequences (UAS) in the promoter, fused to the reporter gene β-glucuronidase (GUS). In an alternate approach, sequences to be analyzed for activation ability were fused to another transcription factor protein, and the GAL4 DBD was fused to a second protein that interacts with the first protein, so that transcriptional activation occurs upon the interaction of the two proteins.

Example IV. Analysis of the EDLL Domain as a Transcription Activator

To analyze the function of the EDLL motif, a 24 amino acid peptide comprising the EDLL motif from AtERF98 (G1792) was fused with the GAL4 DNA binding domain (GAL4DBD or GD in the text) from yeast. The GAL4 DBD:EDLL fusion protein (GD:EDLL in the text) was co-transfected into plant protoplasts along with a reporter gene (in this case β-glucuronidase, GUS) containing GAL4 binding sequences (UAS) in the promoter (FIG. 2). The chimeric protein, (“GD-EDLL” in this figure), when expressed in plant protoplasts, induced GUS expression to approximately the same extent as a fusion of the GAL4 DBD with the well-characterized VP16 activation domain (GD-VP16), whereas the GAL4 DBD alone (GD) which lacks any activation sequences could not induce GUS expression. When the conserved hydrophobic leucine residues were changed to valine (“EDLLm”), the activation potential of EDLL motif was significantly compromised. Two copies of the EDLL motif were also shown to be effective in transcription activation (FIG. 3; “GD:EDLL(2×)”)

Results presented in FIG. 4 demonstrated the utility of the EDLL domain in activating transcription by way of a transcription regulatory polypeptide (G481, an NF-Y or CCAAT-binding transcription factor) unrelated to the sequence from which the EDLL domain was derived (AtERF98, an AP2 family transcription factor. These results demonstrated that the EDLL motif can confer transcriptional activation function to a plant transcription factor or other sequence of interest lacking activation capacity. Furthermore, the EDLL motif is also active even if the protein is not binding DNA directly (G481: EDLL alone can not bind DNA; data not shown) but is recruited to the DNA via interaction with another DNA binding protein (for example, GD:G483 or GD:G715), demonstrating that it can function in larger transcriptional complexes.

Results presented in FIG. 5 demonstrated that the EDLL motif can function to convert a transcriptional repressor into a transcriptional activator. G400, SEQ ID NO: 116, is a homeodomain-leucine zipper (HD-Zip) transcription factor that contains a repression domain termed an EAR domain (Ciarbelli et al. (2008) Plant Mol Biol. 68: 465-478). This protein binds to the promoter of another HD-Zip gene (prG398; SEQ ID NO: 99), but does not activate transcription (Myc:G400; encoded by SEQ ID NO: 128) relative to a non-specific control construct (CAT). Addition of the EDLL domain to this transcription factor (G400:EDLL:Myc; encoded by SEQ ID NO: 130) produced significant activation of prG398:GUS fusion construct, even though the native repression domain was still present. Addition of the EDLL domain to a variant of G400 with the EAR domain mutated (G400EAR:EDLL:Myc; encoded by SEQ ID NO: 98) produced even greater activation of the reporter fusion.

The EDLL motif was fused to various transcription factors and transformed into Arabidopsis plants. For example, the AP2 transcription factor G28, which when overexpressed produces plants that are smaller in size, darker green, later flowering and more disease resistant than comparable control plants, was fused to the EDLL domain (SEQ ID NO: 100) and transformed into Arabidopsis plants under the control of the constitutive 35S promoter (SEQ ID NO: 115) and the pathogen-inducible promoter prAT1G35230 (SEQ ID NO: 114). Plants from a T1 population of 35S::G28:EDLL (SEQ ID NO: 100) plants were generally smaller and darker green than those in a comparable T1 population of 35S::G28 plants, indicating that the EDLL fusion has greater potency than G28 alone. An enhanced dark green phenotype, as exhibited by the 35S::G28:EDLL lines could be indicative of enhanced photosynthetic potential, which could lead to enhanced yield. These plants as well as plants expressing G28:EDLL under prAT1G35230 will be assayed for disease resistance, and we anticipate that the G28:EDLL fusions will produce stronger disease resistance than the unmodified G28 transcription factor. Similarly, a number of transcription factors that provide abiotic stress tolerance (e.g. drought tolerance) when overexpressed have been modified by addition of the EDLL domain and transformed into Arabidopsis under the constitutive 35S promoter, the abiotic stress inducible RD29a promoter (SEQ ID NO: 111), or the drought inducible prAt5G43840 (SEQ ID NO: 112) and prAT5G52300 (SEQ ID NO: 113) promoters. These include the NF-YB transcription factors G481 (SEQ ID NO: 73, encoded by the G481:EDLL:cMyc fusion SEQ ID NO: 96) and G482 (SEQ ID NO: 131, encoded by the G482:EDLL fusion SEQ ID NO: 109), the WRKY transcription factor G1274 (SEQ ID NO: 132, encoded by the 35S::G1274:EDLL fusion SEQ ID NO: 101), the RAV transcription factor G867 (SEQ ID NO: 133, encoded by the prAt5G43840::G867:EDLL fusion SEQ ID NO: 102), the MADS transcription factor G1760 (SEQ ID NO: 134, encoded by the prAt5G43840::G1760:EDLL fusion SEQ ID NO: 104), the AP2 transcription factors G913 (SEQ ID NO: 135) and G912 (SEQ ID NO: 136), and the bHLH transcription factor G2932 (SEQ ID NO: 137). In addition, we fused the EDLL domain to two transcription factors that interact with the NF-YB transcription factor G481 and which could potentially be recruited to the NF-Y complex: the NF-YA transcription factor G926 (SEQ ID NO: 138) and the NF-YC transcription factor G715 (SEQ ID NO: 139). We anticipate that these transcription factors with the addition of the EDLL domain will produce more potent stress tolerance or confer a greater enhancement of yield potential than the comparable unmodified transcription factors.

The EDLL motif will be assayed as a fusion to other DNA binding proteins (transcription factors and co-regulators in plants). These EDLL chimeric fusion proteins will be transformed into Arabidopsis and other crop plants. Various promoters such as constitutive promoters (for example, Cauliflower Mosaic Virus 35S, rice actin) tissue-specific promoters, and the native promoters of the transcription factors to be tested will be used for the expression of chimeric proteins. It is expected that these chimeric proteins will confer various beneficial agronomic traits, including, for example, increased yield, improved water deficit tolerance, enhanced tolerance to hyperosmotic stress, enhanced tolerance to low or high temperatures, increased photosynthetic efficiency, increased disease resistance, earlier or delayed flowering time, and/or enhanced quantity or quality of proteins in seeds and tubers, relative to a control plant or relative to a plant comparably transformed with the DNA binding protein without the EDLL chimeric fusion.

Example V. Analysis of EDLL Domains from Diverse Plant Species

Peptides comprising EDLL motifs from soy, Medicago, rice, and maize, as well as the EDLL motif from an Arabidopsis paralog of AtERF98 (G30), were synthesized and cloned in frame with the yeast GAL4DNA binding domain (GD). The activation function of these sequences was analyzed as described for the AtERF98 EDLL domain in Example III, and all of these sequences produced transcriptional activation of the reporter gene (FIG. 6).

Thus, the EDLL motif is conserved in diverse plant genera including eudicots and monocots. The number of sequences described herein, for example, in Table 1 or the sequence listing, represent a practical sampling of a considerable number of sequence species. Between the eudicots soy, alfalfa, and Arabidopsis, and the monocots rice, wheat, and maize, are a very large number of plant species and their related sequences. There are about 199,350 eudicot plant species (Thorne (2002) Taxon 51: 511-512) that can produce G1792 clade member proteins evolutionarily more closely related to SEQ ID NO: 37 than to EDLL domains from the rice or maize orthologs. As shown below, EDLL domains from both monocot and dicot species have retained function as well as structure. These functionally-related sequences indicate that a considerable majority, if not all or almost all, of the plant species between Arabidopsis and monocot species will have conserved their EDLL domain sequences and associated function. Many orthologous monocot-derived sequences (there are about 59,300 monocot species; Thorne (2002) supra) should also retain similar functions; it seems unlikely that rice, wheat and maize are the only monocot plants to have retained orthologous EDLL domains after 130 to 240 million years of evolution (the generally accepted span from the monocot-eudicot divergence). Thus, a very large number of functional EDLL domain sequences can be readily found in plant species that lie in intermediate positions on the evolutionary tree.

The EDLL motif will be isolated from other crop orthologs such as wheat using similar approaches. These motifs from various crop orthologs will be analyzed using approaches described in Example III. We are also intending to isolate EDLL motifs by using genome sequencing, cDNA and genomic library screening or by RT-PCR using degenerate oligos from varieties such as Sorghum, Miscanthus and others plants where sequence information is not available. The motifs from these species will be analyzed similarly for their activation potential. Additionally, artificial EDLL motifs may be designed and created by synthesis and cloned in frame with yeast GAL4DNA binding domain (GD) and analyzed similarly for their activation potential.

Example VI. Activation of Various Transcription Regulatory Polypeptides with the EDLL Domain

In addition to the transcription factors described above that were modified by addition of an EDLL domain, it can be anticipated that other transcription factors or other polypeptides of interest could be similarly modified. Appendix A provides further examples of A. thaliana transcription factor and other protein sequences that can be modified by fusion to one or more of the EDLL domains found in Table 1, or in variants thereof, as provided herein or manufactured using methods known in the art. Homologs of these transcription factor and other sequences may also be so modified. In this regard, “homolog” is defined as a gene encoding a particular protein sequence from a eukaryotic organism including Arabidopsis thaliana (in the case or paralogs) or other than A. thaliana (in the case or orthologs): (a) that when compared to the set of protein sequences encoded by the A. thaliana genome, has a similarity equal to or better than the “Minimum Similarity Requirement” (defined below); and (b) that is more similar to a gene in Appendix A than it is to any other protein sequence encoded by the A. thaliana genome. Similarity may be measured using the BLASTP algorithm available from the National Center for Biotechnology Information with, for example, the default parameters of the software program. The “Minimum Similarity Requirement” for a match may be defined as a high-scoring segment pair (HSP(s)) of bit score fifty (50) or better.

Example VII. Practical Benefits of Using the EDLL Domain for Enhancing Transcription

The EDLL domain is a new transcriptional activation domain identified from a plant protein. It is highly active when fused with different class of DBD proteins from plants and yeast and has activation potential comparable to widely used VP16 activation domain, derived from Herpes simplex. The domain has many practical benefits. Some of these are described below:

-   1. Small size. Unlike other known activation domains such as VP16     and GAL4, EDLL is relatively small in size and fusing of a peptide     this small to any protein has a lower chance of altering the native     conformation of a fusion protein. Further deletion analysis to     determine the minimum region required for transcriptional activation     is in progress. -   2. Plant-derived. The EDLL domain is the first strong     transcriptional activation domain from a plant species to be     well-characterized. Transcription factors containing this domain are     also present in many other plant species including useful crop     varieties like rice, maize, soybean and alfalfa. The EDLL domain     from these crops, or from other plant species, can be fused with     transcription factors isolated from same species, or other plant     species, and can be used for enhanced induction of any target genes     in those crop varieties. This approach affords enhanced activation     of TF targets while avoiding contamination of the crop genome with     expressed genetic materials derived from outside of the plant     kingdom. -   3. Strong activation potential. Based on our experimental analyses     described above, the EDLL motif has activation potential at least     similar to, if not higher than most characterized activation domains     in the literature, for example, VP16. -   4. Optimization of activity. The strength of the EDLL domain     activation potential may be fine-tuned by modifying one or more     amino acid residues in the domain, for example, through the use of     site-specific mutagenesis. In this manner, the ability of a specific     transcription factor to activate transcription of its target genes     can be adjusted to a greater or lesser extent with the use of a     native or modified EDLL domain. -   5. Broad activity. The EDLL motif is active on proteins isolated     from both plants and yeast, and it is also active when targeted to     the promoter of the desired gene by a protein with a DNA binding     motif or through a protein-protein interaction motif. These     properties of the EDLL motif make it a useful tool for targeted     induction of desired genes in plants, and also it can be used for     making research tools for protein-DNA and protein-protein     interactions in bacteria, yeast, plants and animals. -   6. Suppression of repression. The EDLL motif will be used to convert     a repressor protein to an activator: We have shown experimentally     (see, for example, the description of FIG. 4, above) that the NF-YB     protein G481, which alone has no transcriptional activation     capacity, can be converted into an activator by fusing it to an EDLL     motif. Overexpression of G481 protein in Arabidopsis produces     down-regulation of the flowering modulator gene FT, which in turn     causes delay in flowering. The G481:EDLL chimeric protein is being     expressed in plants and it is anticipated that the chimeric protein     which functions as an activator will accelerate flowering. The EDLL     motif is also being fused to other subunits of the NF-Y protein     complex, and to other transcription factors and co-regulators of     various physiological and developmental pathways in plants. -   7. Agronomic potential. The EDLL activation domain has a wide range     of agronomic potential. The expression of any gene can be regulated     by fusing the EDLL motif to a sequence-specific DNA binding protein     or various co-regulators capable of binding to the promoter of that     target gene. Various developmental, physiological and environmental     pathways could be modulated by these means, including, cell division     and growth, photosynthesis, shade avoidance, drought and temperature     tolerance, and disease resistance, with the goal of enhancing yield     in crop varieties. It can also be used for enhancing the     accumulation and quality of protein in plants like cassava and other     tuber forming varieties routinely being used as major food crops in     African countries, or modifying the accumulation of carbohydrates,     including starches and sugars, in important food or fuel crops     including but not limited to corn, rice, wheat, sorghum, sugar cane,     miscane (a sugar cane×Miscanthus hybrid cross) and Miscanthus. In     particular, we expect that the EDLL motif might be used to optimize     transcription factors when used in combination with an inducible     promoter, such as RD29A which is responsive to drought or cold. For     example, certain transcription factors from the AP2 family, such as     the CBF group, which confer abiotic stress tolerance when     constitutively overexpressed, can also result in stunting;     regulation of chimeric fusions of these AP2 family transcription     factors with an EDLL motif combined with a tissue specific or     inducible promoter provides a means of obtaining an enhanced crop     without substantial negative phenotypes.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The present invention is not limited by the specific embodiments described herein. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the Claims. Modifications that become apparent from the foregoing description and accompanying figures fall within the scope of the following Claims.

APPENDIX A AGI Number TF Family AGI Number TF Family AGI Number TF Family AGI Number TF Family AT5G06250 ABI3/VP-1 AT4G17695 GARP AT5G13790 MADS AT2G44730 TH AT4G32010 ABI3/VP-1 AT5G16560 GARP AT1G60920 MADS AT1G54060 TH AT4G21550 ABI3/VP-1 AT4G37180 GARP AT1G60880 MADS AT3G10000 TH AT2G30470 ABI3/VP-1 AT4G16110 GARP AT1G59810 MADS AT3G24490 TH AT1G28300 ABI3/VP-1 AT2G01060 GARP AT1G48150 MADS AT1G21200 TH AT3G11580 ABI3/VP-1 AT5G05090 GARP AT1G69120 MADS AT1G76890 TH AT2G36080 ABI3/VP-1 AT4G31920 GARP AT4G18960 MADS AT1G33240 TH AT2G46870 ABI3/VP-1 AT1G49560 GARP AT4G24540 MADS AT1G16070 TUBBY AT2G33720 ABI3/VP-1 AT2G01760 GARP AT5G40120 MADS AT1G25280 TUBBY AT3G26790 ABI3/VP-1 AT3G24120 GARP AT5G62165 MADS AT5G18680 TUBBY AT4G01500 ABI3/VP-1 AT4G28610 GARP AT5G65330 MADS AT1G47270 TUBBY AT1G01030 ABI3/VP-1 AT3G04030 GARP AT5G41200 MADS AT1G43640 TUBBY AT3G61970 ABI3/VP-1 AT1G68670 GARP AT5G40220 MADS AT1G61940 TUBBY AT3G24650 ABI3/VP-1 AT1G69580 GARP AT5G39810 MADS AT1G53320 TUBBY AT3G19130 ACBF-like AT1G49190 GARP AT5G39750 MADS AT3G06380 TUBBY AT5G19350 ACBF-like AT2G20400 GARP AT5G38740 MADS AT2G18280 TUBBY AT1G47500 ACBF-like AT1G13300 GARP AT5G27580 MADS AT2G47900 TUBBY AT5G54900 ACBF-like AT2G02060 GARP AT5G27090 MADS AT1G76900 TUBBY AT1G49600 ACBF-like AT5G18240 GARP AT5G27070 MADS AT1G14410 WHY AT1G11650 ACBF-like AT5G29000 GARP AT5G27050 MADS AT1G71260 WHY AT1G47490 ACBF-like AT4G04580 GARP AT5G26950 MADS AT2G02740 WHY AT4G27000 ACBF-like AT5G45580 GARP AT5G04640 MADS AT2G25000 WRKY AT3G23300 AKR AT2G03500 GARP AT4G11250 MADS AT4G31550 WRKY AT5G06050 AKR AT2G40260 GARP AT3G18650 MADS AT1G13960 WRKY AT5G14430 AKR AT2G42660 GARP AT2G42830 MADS AT4G31800 WRKY AT1G26850 AKR AT2G20570 GARP AT3G02310 MADS AT2G04880 WRKY AT2G43200 AKR AT4G13640 GARP AT2G03710 MADS AT5G41570 WRKY AT2G45750 AKR AT3G10760 GARP AT5G15800 MADS AT2G44745 WRKY AT2G34300 AKR AT5G42630 GARP AT3G58780 MADS AT4G12020 WRKY AT1G29470 AKR AT1G25550 GARP AT1G26310 MADS AT3G01970 WRKY AT5G61230 AKR AT1G67710 GARP AT5G20240 MADS AT4G30935 WRKY AT1G19430 AKR AT2G25180 GARP AT3G54340 MADS AT2G30250 WRKY AT5G64030 AKR AT2G27070 GARP AT4G11880 MADS AT5G07100 WRKY AT5G04060 AKR AT5G59570 GARP AT3G61120 MADS AT4G01250 WRKY AT3G12360 AKR AT3G16857 GARP AT2G34440 MADS AT5G26170 WRKY AT5G15500 AKR AT3G62670 GARP AT5G60440 MADS AT5G64810 WRKY AT1G77260 AKR AT3G04450 GARP AT4G36590 MADS AT2G37260 WRKY AT5G45110 AKR AT3G12730 GARP AT1G01530 MADS AT3G58710 WRKY AT1G78240 AKR AT3G13040 GARP AT2G03060 MADS AT3G04670 WRKY AT1G14480 AKR AT3G25790 GARP AT1G77080 MADS AT3G56400 WRKY AT3G51070 AKR AT1G14600 GARP AT5G10140 MADS AT4G24240 WRKY AT3G57130 AKR AT3G46640 GARP AT5G65080 MADS AT5G43290 WRKY AT4G18030 AKR AT5G58080 GARP AT5G65070 MADS AT2G46130 WRKY AT1G04430 AKR AT5G07210 GARP AT5G65060 MADS AT2G46400 WRKY AT4G14365 AKR AT5G49240 GARP AT1G77950 MADS AT4G26640 WRKY AT5G66055 AKR AT1G32240 GARP AT1G77980 MADS AT5G49520 WRKY AT2G39750 AKR AT5G06800 GARP AT1G47760 MADS AT5G45260 WRKY AT1G64280 AKR AT3G19070 GARP AT3G05860 MADS AT4G01720 WRKY AT2G24600 AKR AT2G06020 GARP AT1G65360 MADS AT2G21900 WRKY AT5G54700 AKR AT4G18020 GARP AT1G65330 MADS AT4G23810 WRKY AT5G54710 AKR AT2G40970 GARP AT5G26870 MADS AT4G23550 WRKY AT2G31820 AKR AT4G36620 GATA/Zn AT5G27130 MADS AT4G26440 WRKY AT5G50140 AKR AT5G47140 GATA/Zn AT5G55690 MADS AT5G45050 WRKY AT1G03670 AKR AT3G51080 GATA/Zn AT5G51870 MADS AT5G22570 WRKY AT4G03440 AKR AT2G28340 GATA/Zn AT5G51860 MADS AT2G23320 WRKY AT4G03450 AKR AT4G32890 GATA/Zn AT2G24840 MADS AT5G52830 WRKY AT4G03460 AKR AT5G25830 GATA/Zn AT5G58890 MADS AT2G34830 WRKY AT5G40160 AKR AT5G66320 GATA/Zn AT1G29962 MADS AT4G39410 WRKY AT4G00750 AKR AT4G26150 GATA/Zn AT3G66656 MADS AT5G46350 WRKY AT4G19660 AKR AT2G18380 GATA/Zn AT1G17310 MADS AT2G47260 WRKY AT1G31850 AKR AT3G54810 GATA/Zn AT3G30260 MADS AT4G18170 WRKY AT4G19120 AKR AT5G56860 GATA/Zn AT1G31630 MADS AT1G62300 WRKY AT1G33170 AKR AT4G17570 GATA/Zn AT1G31640 MADS AT5G24110 WRKY AT4G10440 AKR AT5G49300 GATA/Zn AT5G06500 MADS AT4G11070 WRKY AT2G03480 AKR AT3G21175 GATA/Zn AT1G22130 MADS AT4G22070 WRKY AT4G00740 AKR AT3G50870 GATA/Zn AT1G54760 MADS AT1G55600 WRKY AT2G41370 AKR AT3G06740 GATA/Zn AT1G60040 MADS AT1G30650 WRKY AT4G26120 AKR AT4G24470 GATA/Zn AT2G22540 MADS AT2G30590 WRKY AT2G40280 AKR AT1G08000 GATA/Zn AT5G65050 MADS AT3G01080 WRKY AT2G03430 AKR AT3G16870 GATA/Zn AT2G26880 MADS AT1G64000 WRKY AT2G47450 AKR AT1G51600 GATA/Zn AT5G48670 MADS AT1G69810 WRKY AT4G10720 AKR AT1G08010 GATA/Zn AT3G24500 MBFL AT1G18860 WRKY AT5G54610 AKR AT3G45170 GATA/Zn AT3G58680 MBFL AT1G68150 WRKY AT2G01680 AKR AT2G45050 GATA/Zn AT2G42680 MBFL AT1G29280 WRKY AT5G60070 AKR AT4G34680 GATA/Zn AT5G66840 MISC AT1G69310 WRKY AT1G05640 AKR AT3G24050 GATA/Zn AT4G32551 MISC AT2G40740 WRKY AT1G10340 AKR AT4G36240 GATA/Zn AT2G32700 MISC AT5G28650 WRKY AT4G03470 AKR AT3G60530 GATA/Zn AT1G43850 MISC AT1G66550 WRKY AT4G03480 AKR AT1G14685 GBP AT4G25520 MISC AT1G66560 WRKY AT4G03490 AKR AT1G68120 GBP AT1G48050 MISC AT5G01900 WRKY AT4G03500 AKR AT2G01930 GBP AT1G73230 MISC AT1G29860 WRKY AT4G05040 AKR AT5G42520 GBP AT1G17880 MISC AT5G15130 WRKY AT4G14400 AKR AT4G38910 GBP AT5G53060 MISC AT5G13080 WRKY AT4G14390 AKR AT2G21240 GBP AT5G23540 MISC AT1G66600 WRKY AT1G34050 AKR AT2G35550 GBP AT1G71230 MISC AT1G80590 WRKY AT3G54990 AP2 AT5G10450 GF14 AT5G44350 MISC AT3G62340 WRKY AT1G22190 AP2 AT5G16050 GF14 AT4G20880 MISC AT5G56270 WRKY AT5G25190 AP2 AT2G42590 GF14 AT3G43340 MISC AT2G24570 WRKY AT1G15360 AP2 AT1G22300 GF14 AT4G16420 MISC AT1G80840 WRKY AT1G25560 AP2 AT1G78220 GF14 AT5G09210 MISC AT2G40750 WRKY AT5G51990 AP2 AT1G34760 GF14 AT5G41580 MISC AT4G04450 WRKY AT2G39250 AP2 AT1G78300 GF14 AT1G08910 MISC AT2G38470 WRKY AT5G52020 AP2 AT5G38480 GF14 AT5G08550 MISC AT2G03340 WRKY AT5G07580 AP2 AT1G35160 GF14 AT1G22920 MISC AT1G23420 YABBY AT4G18450 AP2 AT4G09000 GF14 AT5G61850 MISC AT4G00180 YABBY AT2G38340 AP2 AT5G65430 GF14 AT3G07740 MISC AT1G69180 YABBY AT1G77200 AP2 AT3G02520 GF14 AT4G25515 MISC AT2G26580 YABBY AT5G53290 AP2 AT2G10450 GF14 AT5G62090 MISC AT2G45190 YABBY AT5G61600 AP2 AT3G52910 GRF-like AT3G13000 NAC AT1G08465 YABBY AT5G65130 AP2 AT2G06200 GRF-like AT1G32770 NAC AT2G40110 YIP AT1G78080 AP2 AT4G37740 GRF-like AT1G65910 NAC AT3G55890 YIP AT3G15210 AP2 AT3G13960 GRF-like AT4G35580 NAC AT4G27740 YIP AT2G47520 AP2 AT4G24150 GRF-like AT2G02450 NAC AT4G27745 YIP AT5G11190 AP2 AT2G42040 GRF-like AT1G01720 NAC AT5G53940 YIP AT5G18560 AP2 AT2G45480 GRF-like AT5G08790 NAC AT3G11230 YIP AT5G17430 AP2 AT2G22840 GRF-like AT3G10490 NAC AT3G08990 YIP AT1G19210 AP2 AT5G53660 GRF-like AT3G15500 NAC AT5G44160 Z-C2H2 AT5G18450 AP2 AT2G36400 GRF-like AT3G10480 NAC AT5G43170 Z-C2H2 AT5G10510 AP2 AT2G23760 HB AT2G24430 NAC AT5G04340 Z-C2H2 AT1G64380 AP2 AT5G44180 HB AT1G77450 NAC AT2G41940 Z-C2H2 AT2G33710 AP2 AT4G32880 HB AT3G04060 NAC AT5G48890 Z-C2H2 AT5G19790 AP2 AT2G34710 HB AT3G29035 NAC AT1G24625 Z-C2H2 AT3G50260 AP2 AT1G30490 HB AT4G36160 NAC AT5G25160 Z-C2H2 AT5G60120 AP2 AT4G17710 HB AT5G64060 NAC AT5G14010 Z-C2H2 AT3G23240 AP2 AT1G79840 HB AT5G63790 NAC AT1G80730 Z-C2H2 AT4G36900 AP2 AT4G00730 HB AT5G17260 NAC AT5G57520 Z-C2H2 AT3G11020 AP2 AT1G05230 HB AT1G02230 NAC AT1G66140 Z-C2H2 AT1G33760 AP2 AT4G21750 HB AT1G02250 NAC AT1G10480 Z-C2H2 AT3G25890 AP2 AT5G60690 HB AT1G02220 NAC AT1G67030 Z-C2H2 AT1G53170 AP2 AT5G65310 HB AT5G61430 NAC AT4G17810 Z-C2H2 AT5G57390 AP2 AT4G16780 HB AT5G24590 NAC AT4G27240 Z-C2H2 AT2G46310 AP2 AT5G47370 HB AT5G04400 NAC AT3G01030 Z-C2H2 AT2G25820 AP2 AT4G17460 HB AT1G02210 NAC AT3G02790 Z-C2H2 AT5G47220 AP2 AT3G60390 HB AT4G28500 NAC AT5G52010 Z-C2H2 AT1G50640 AP2 AT2G44910 HB AT1G26870 NAC AT5G66730 Z-C2H2 AT5G47230 AP2 AT5G06710 HB AT3G44290 NAC AT1G51220 Z-C2H2 AT4G06746 AP2 AT4G37790 HB AT2G18060 NAC AT5G60470 Z-C2H2 AT1G53910 AP2 AT2G22800 HB AT5G62380 NAC AT4G02670 Z-C2H2 AT1G51190 AP2 AT1G20710 HB AT1G60350 NAC AT5G03150 Z-C2H2 AT5G25390 AP2 AT1G70920 HB AT1G60340 NAC AT3G23130 Z-C2H2 AT2G35700 AP2 AT1G75430 HB AT1G60380 NAC AT2G28200 Z-C2H2 AT2G20880 AP2 AT5G46010 HB AT1G60300 NAC AT4G16610 Z-C2H2 AT1G36060 AP2 AT1G20700 HB AT1G60280 NAC AT2G37430 Z-C2H2 AT3G60490 AP2 AT1G46480 HB AT4G01520 NAC AT3G46090 Z-C2H2 AT3G20310 AP2 AT5G53980 HB AT4G01540 NAC AT5G59820 Z-C2H2 AT5G67190 AP2 AT4G25530 HB AT4G01550 NAC AT5G67450 Z-C2H2 AT4G11140 AP2 AT4G08150 HB AT1G32870 NAC AT4G31420 Z-C2H2 AT5G51190 AP2 AT2G17950 HB AT1G33060 NAC AT1G14580 Z-C2H2 AT5G67180 AP2 AT3G18010 HB AT3G15510 NAC AT3G19580 Z-C2H2 AT1G75490 AP2 AT5G66700 HB AT5G66300 NAC AT3G45260 Z-C2H2 AT5G61890 AP2 AT5G59340 HB AT2G27300 NAC AT5G05120 Z-C2H2 AT4G37750 AP2 AT1G69780 HB AT1G56010 NAC AT5G43540 Z-C2H2 AT5G25810 AP2 AT1G73360 HB AT3G49530 NAC AT2G42410 Z-C2H2 AT2G40350 AP2 AT3G03260 HB AT1G52880 NAC AT2G37740 Z-C2H2 AT2G40340 AP2 AT1G28420 HB AT4G27410 NAC AT2G26940 Z-C2H2 AT2G36450 AP2 AT1G75410 HB AT5G39820 NAC AT5G54360 Z-C2H2 AT2G31230 AP2 AT2G28610 HB AT5G56620 NAC AT5G54340 Z-C2H2 AT4G34410 AP2 AT2G27990 HB AT5G64530 NAC AT4G04404 Z-C2H2 AT4G27950 AP2 AT2G35940 HB AT5G14000 NAC AT5G61470 Z-C2H2 AT2G20350 AP2 AT4G04890 HB AT1G62700 NAC AT1G02040 Z-C2H2 AT2G22200 AP2 AT2G01500 HB AT2G43000 NAC AT2G17180 Z-C2H2 AT3G57600 AP2 AT5G17810 HB AT1G34180 NAC AT3G13810 Z-C2H2 AT1G12980 AP2 AT5G45980 HB AT1G01010 NAC AT2G45120 Z-C2H2 AT5G43410 AP2 AT2G33880 HB AT3G18400 NAC AT3G20880 Z-C2H2 AT5G64750 AP2 AT4G35550 HB AT3G17730 NAC AT3G23140 Z-C2H2 AT3G20840 AP2 AT5G46880 HB AT3G12910 NAC AT2G28710 Z-C2H2 AT3G23230 AP2 AT5G05770 HB AT3G04420 NAC AT2G01940 Z-C2H2 AT3G23220 AP2 AT1G34650 HB AT5G46590 NAC AT3G50700 Z-C2H2 AT4G32800 AP2 AT1G23380 HB AT1G32510 NAC AT3G58070 Z-C2H2 AT4G28140 AP2 AT2G27220 HB AT1G34190 NAC AT3G53600 Z-C2H2 AT5G50080 AP2 AT2G16400 HB AT1G76420 NAC AT3G53820 Z-C2H2 AT5G67010 AP2 AT4G34610 HB AT3G03200 NAC AT1G68130 Z-C2H2 AT5G67000 AP2 AT2G36610 HB AT1G79580 NAC AT3G10470 Z-C2H2 AT5G65510 AP2 AT1G52150 HB AT3G56560 NAC AT1G13290 Z-C2H2 AT3G16770 AP2 AT2G18550 HB AT3G56530 NAC AT1G08290 Z-C2H2 AT2G41710 AP2 AT2G32370 HB AT3G56520 NAC AT3G09290 Z-C2H2 AT4G36920 AP2 AT2G01430 HB AT5G14490 NAC AT3G57670 Z-C2H2 AT1G28360 AP2 AT1G26960 HB AT3G44350 NAC AT3G46070 Z-C2H2 AT3G25730 AP2 AT4G03250 HB AT3G61910 NAC AT3G60580 Z-C2H2 AT4G13620 AP2 AT5G02030 HB AT1G71930 NAC AT5G56200 Z-C2H2 AT1G49120 AP2 AT3G01220 HB AT3G04070 NAC AT3G29340 Z-C2H2 AT1G79700 AP2 AT3G03660 HB AT1G28470 NAC AT1G26610 Z-C2H2 AT3G61630 AP2 AT1G19700 HB AT4G10350 NAC AT2G02070 Z-C2H2 AT1G21910 AP2 AT1G62990 HB AT3G15170 NAC AT2G02080 Z-C2H2 AT1G72570 AP2 AT5G52170 HB AT1G33280 NAC AT2G23740 Z-C2H2 AT1G28370 AP2 AT1G17920 HB AT3G04430 NAC AT2G15740 Z-C2H2 AT1G12890 AP2 AT3G11260 HB AT3G10500 NAC AT2G18490 Z-C2H2 AT1G63030 AP2 AT1G62360 HB AT1G03490 NAC AT5G10970 Z-C2H2 AT1G16060 AP2 AT1G70510 HB AT1G19040 NAC AT1G55110 Z-C2H2 AT1G72360 AP2 AT5G11060 HB AT1G64105 NAC AT5G06070 Z-C2H2 AT1G71450 AP2 AT5G25220 HB AT5G50820 NAC AT5G03510 Z-C2H2 AT1G71520 AP2 AT4G32040 HB AT3G04410 NAC AT5G01860 Z-C2H2 AT2G44940 AP2 AT4G32980 HB AT5G18300 NAC AT5G22890 Z-C2H2 AT1G77640 AP2 AT5G41410 HB AT5G18270 NAC AT3G46080 Z-C2H2 AT2G44840 AP2 AT4G36870 HB AT5G39610 NAC AT5G06650 Z-C2H2 AT1G71130 AP2 AT4G29940 HB AT2G33480 NAC AT4G35610 Z-C2H2 AT1G44830 AP2 AT3G19510 HB AT5G07680 NAC AT4G26030 Z-C2H2 AT1G28160 AP2 AT1G27050 HB AT1G60240 NAC AT1G34370 Z-C2H2 AT1G80580 AP2 AT2G46680 HB AT5G39690 NAC AT1G26590 Z-C2H2 AT3G16280 AP2 AT4G36740 HB AT5G41090 NAC AT1G03840 Z-C2H2 AT5G11590 AP2 AT5G15150 HB AT4G29230 NAC AT5G42640 Z-C2H2 AT5G21960 AP2 AT3G01470 HB AT3G01600 NAC AT5G27880 Z-C2H2 AT2G23340 AP2 AT2G22430 HB AT3G55210 NAC AT5G22990 Z-C2H2 AT1G12630 AP2 AT5G17320 HB AT5G09330 NAC AT3G49930 Z-C2H2 AT1G12610 AP2 AT3G61890 HB AT1G25580 NAC AT1G68360 Z-C2H2 AT1G06160 AP2 AT4G40060 HB AT5G22290 NAC AT1G25250 Z-C2H2 AT1G01250 AP2 AT3G61150 HB AT5G22380 NAC AT1G43860 Z-C2H2 AT5G07310 AP2 AT5G03790 HB AT4G28530 NAC AT2G29660 Z-C2H2 AT4G16750 AP2 AT3G19860 HLH/MYC AT2G17040 NAC AT1G30970 Z-C2H2 AT1G25470 AP2 AT3G47640 HLH/MYC AT4G17980 NAC AT5G15480 Z-C2H2 AT1G51120 AP2 AT1G02340 HLH/MYC AT5G53950 NAC AT5G54630 Z-C2H2 AT1G50680 AP2 AT1G03040 HLH/MYC AT1G61110 NAC AT5G16470 Z-C2H2 AT4G17500 AP2 AT1G51070 HLH/MYC AT1G69490 NAC AT2G27100 Z-C2H2 AT1G24590 AP2 AT5G54680 HLH/MYC AT5G13180 NAC AT4G12240 Z-C2H2 AT1G22985 AP2 AT3G23210 HLH/MYC AT1G12260 NAC AT4G35700 Z-C2H2 AT4G39780 AP2 AT3G59060 HLH/MYC AT5G04410 NAC AT1G34790 Z-C2H2 AT5G61590 AP2 AT2G22770 HLH/MYC AT1G52890 NAC AT3G29340 Z-C2H2 AT4G25480 AP2 AT5G57150 HLH/MYC AT1G54330 NAC AT4G16845 Z-C2H2 AT4G25470 AP2 AT1G29950 HLH/MYC AT2G46770 NAC AT5G51230 Z-C2H2 AT4G25490 AP2 AT3G23690 HLH/MYC AT5G56780 OTHER AT2G35670 Z-C2H2 AT3G14230 AP2 AT1G05805 HLH/MYC AT4G26170 OTHER AT1G11490 Z-C2H2 AT4G31060 AP2 AT5G43175 HLH/MYC AT4G27330 OTHER AT1G75710 Z-C2H2 AT5G05410 AP2 AT5G61270 HLH/MYC AT5G35770 OTHER AT4G35280 Z-C2H2 AT1G22810 AP2 AT3G05800 HLH/MYC AT5G21030 PAZ AT1G27730 Z-C2H2 AT4G17490 AP2 AT1G27740 HLH/MYC AT2G27880 PAZ AT1G49900 Z-C2H2 AT1G04370 AP2 AT3G57800 HLH/MYC AT1G48410 PAZ AT1G02030 Z-C2H2 AT1G46768 AP2 AT3G50330 HLH/MYC AT2G27040 PAZ AT2G24500 Z-C2H2 AT5G44210 AP2 AT1G69010 HLH/MYC AT1G69440 PAZ AT2G19810 Z-C3H AT1G03800 AP2 AT1G68920 HLH/MYC AT2G32940 PAZ AT4G29190 Z-C3H AT5G13910 AP2 AT1G68810 HLH/MYC AT5G43810 PAZ AT2G40140 Z-C3H AT2G40220 AP2 AT3G06590 HLH/MYC AT5G08330 PCF AT3G19360 Z-C3H AT1G43160 AP2 AT1G59640 HLH/MYC AT1G58100 PCF AT3G12130 Z-C3H AT1G74930 AP2 AT3G06120 HLH/MYC AT3G47620 PCF AT1G68200 Z-C3H AT1G68550 AP2 AT5G65640 HLH/MYC AT2G37000 PCF AT5G58620 Z-C3H AT1G13260 AP2 AT1G31050 HLH/MYC AT5G23280 PCF AT1G32360 Z-C3H AT5G13330 AP2 AT5G58010 HLH/MYC AT3G27010 PCF AT2G35430 Z-C3H AT4G23750 AP2 AT3G19500 HLH/MYC AT5G51910 PCF AT2G41900 Z-C3H AT2G28550 AP2 AT1G18400 HLH/MYC AT5G41030 PCF AT5G07500 Z-C3H AT1G68840 AP2 AT5G48560 HLH/MYC AT3G45150 PCF AT3G55980 Z-C3H AT3G54320 AP2 AT5G08130 HLH/MYC AT1G72010 PCF AT1G03790 Z-C3H AT1G30330 ARF AT4G37850 HLH/MYC AT2G45680 PCF AT5G44260 Z-C3H AT3G61830 ARF AT4G05170 HLH/MYC AT1G35560 PCF AT5G12850 Z-C3H AT1G34310 ARF AT4G29930 HLH/MYC AT1G69690 PCF AT5G06770 Z-C3H AT5G20730 ARF AT4G28811 HLH/MYC AT2G22300 PCGL AT2G25900 Z-C3H AT1G19220 ARF AT2G40200 HLH/MYC AT5G64220 PCGL AT4G22140 Z-C4HC3 AT1G59750 ARF AT1G30670 HLH/MYC AT4G16150 PCGL AT4G04260 Z-C4HC3 AT5G37020 ARF AT2G43140 HLH/MYC AT5G09410 PCGL AT5G26210 Z-C4HC3 AT2G46530 ARF AT3G25710 HLH/MYC AT1G67310 PCGL AT5G05610 Z-C4HC3 AT1G77850 ARF AT3G17100 HLH/MYC AT3G16940 PCGL AT3G42790 Z-C4HC3 AT4G30080 ARF AT3G20640 HLH/MYC AT2G23380 PCOMB AT3G11200 Z-C4HC3 AT1G34390 ARF AT4G29100 HLH/MYC AT1G02580 PCOMB AT1G14510 Z-C4HC3 AT1G35240 ARF AT1G27660 HLH/MYC AT4G02020 PCOMB AT5G20510 Z-C4HC3 AT1G34410 ARF AT1G05710 HLH/MYC AT1G79020 PCOMB AT2G02470 Z-C4HC3 AT1G35520 ARF AT2G31730 HLH/MYC AT1G16690 PCOMB AT4G39100 Z-C4HC3 AT1G35540 ARF AT2G20100 HLH/MYC AT3G20740 PCOMB AT4G36020 Z-CLDSH AT2G28350 ARF AT1G61660 HLH/MYC AT1G31040 PLATZ AT4G38680 Z-CLDSH AT1G43950 ARF AT5G51790 HLH/MYC AT4G17900 PLATZ AT2G17870 Z-CLDSH AT2G33860 ARF AT5G51780 HLH/MYC AT3G60670 PLATZ AT2G21060 Z-CLDSH AT4G23980 ARF AT5G38860 HLH/MYC AT5G46710 PLATZ AT2G24790 Z-CO-like AT5G62000 ARF AT1G25330 HLH/MYC AT1G21000 PLATZ AT1G28050 Z-CO-like AT1G19850 ARF AT1G49830 HLH/MYC AT1G76590 PLATZ AT4G15248 Z-CO-like AT5G60450 ARF AT1G73830 HLH/MYC AT1G43000 PLATZ AT1G60250 Z-CO-like AT1G34170 ARF AT5G04150 HLH/MYC AT2G27930 PLATZ AT4G39070 Z-CO-like AT1G76110 ARID AT4G25400 HLH/MYC AT2G12646 PLATZ AT2G21320 Z-CO-like AT1G76510 ARID AT4G25410 HLH/MYC AT2G01818 PLATZ AT1G49130 Z-CO-like AT1G20910 ARID AT3G56970 HLH/MYC AT1G32700 PLATZ AT3G21880 Z-CO-like AT2G17410 ARID AT3G56980 HLH/MYC AT1G12860 PMR AT3G21150 Z-CO-like AT1G55650 ARID AT1G12540 HLH/MYC AT2G43440 PMR AT5G15840 Z-CO-like AT1G04880 ARID AT1G71200 HLH/MYC AT2G43270 PMR AT3G07650 Z-CO-like AT3G13350 ARID AT1G62975 HLH/MYC AT2G05600 PMR AT3G02380 Z-CO-like AT1G06280 AS2/LOB AT2G41240 HLH/MYC AT2G02030 PMR AT1G06040 Z-CO-like AT1G67100 AS2/LOB AT5G56960 HLH/MYC AT2G43445 PMR AT5G15850 Z-CO-like AT2G31310 AS2/LOB AT5G65320 HLH/MYC AT2G43260 PMR AT2G33500 Z-CO-like AT2G30340 AS2/LOB AT1G10585 HLH/MYC AT2G42955 PMR AT4G15250 Z-CO-like AT2G30130 AS2/LOB AT4G20970 HLH/MYC AT1G13200 PMR AT1G05290 Z-CO-like AT2G28500 AS2/LOB AT5G43650 HLH/MYC AT1G11270 PMR AT2G47890 Z-CO-like AT2G23660 AS2/LOB AT4G30180 HLH/MYC AT5G61380 PRR AT1G25440 Z-CO-like AT2G19820 AS2/LOB AT2G47270 HLH/MYC AT5G24470 PRR AT1G68520 Z-CO-like AT2G19510 AS2/LOB AT4G38070 HLH/MYC AT2G46790 PRR AT4G10240 Z-CO-like AT1G72980 AS2/LOB AT5G37800 HLH/MYC AT2G46670 PRR AT1G75540 Z-CO-like AT1G68510 AS2/LOB AT5G67110 HLH/MYC AT5G02810 PRR AT4G27310 Z-CO-like AT3G58190 AS2/LOB AT2G31220 HLH/MYC AT5G60100 PRR AT5G24930 Z-CO-like AT3G50510 AS2/LOB AT2G31210 HLH/MYC AT1G26680 REM AT5G48250 Z-CO-like AT3G49940 AS2/LOB AT1G06170 HLH/MYC AT1G49480 REM AT1G73870 Z-CO-like AT3G47870 AS2/LOB AT1G10610 HLH/MYC AT3G53310 REM AT3G21890 Z-CO-like AT3G27940 AS2/LOB AT1G49770 HLH/MYC AT3G06220 REM AT2G31380 Z-CO-like AT3G27650 AS2/LOB AT1G51140 HLH/MYC AT3G46770 REM AT1G68190 Z-CO-like AT3G26660 AS2/LOB AT1G26260 HLH/MYC AT5G09780 REM AT4G38960 Z-CO-like AT3G26620 AS2/LOB AT4G28815 HLH/MYC AT5G66980 REM AT1G78600 Z-CO-like AT3G13850 AS2/LOB AT5G41315 HLH/MYC AT5G60140 REM AT5G57660 Z-CO-like AT3G11090 AS2/LOB AT4G17880 HLH/MYC AT5G60130 REM AT5G54470 Z-CO-like AT3G03760 AS2/LOB AT1G32640 HLH/MYC AT5G57720 REM AT3G50410 Z-Dof AT3G02550 AS2/LOB AT4G00870 HLH/MYC AT5G18090 REM AT2G34140 Z-Dof AT2G45420 AS2/LOB AT4G00480 HLH/MYC AT5G18000 REM AT4G00940 Z-Dof AT2G45410 AS2/LOB AT1G01260 HLH/MYC AT4G33280 REM AT2G28810 Z-Dof AT2G42440 AS2/LOB AT4G00120 HLH/MYC AT4G00260 REM AT5G60200 Z-Dof AT2G42430 AS2/LOB AT4G00050 HLH/MYC AT2G24650 REM AT3G52440 Z-Dof AT2G40470 AS2/LOB AT4G36060 HLH/MYC AT2G24650 REM AT1G07640 Z-Dof AT5G67420 AS2/LOB AT5G23290 HLH/MYC AT2G24680 REM AT2G37590 Z-Dof AT5G66870 AS2/LOB AT4G36540 HLH/MYC AT2G24690 REM AT3G55370 Z-Dof AT5G63090 AS2/LOB AT4G14410 HLH/MYC AT2G24700 REM AT5G60850 Z-Dof AT5G35900 AS2/LOB AT2G42280 HLH/MYC AT4G31690 REM AT1G69570 Z-Dof AT5G06080 AS2/LOB AT4G02590 HLH/MYC AT4G31680 REM AT3G21270 Z-Dof AT4G37540 AS2/LOB AT4G36930 HLH/MYC AT4G31660 REM AT1G51700 Z-Dof AT4G22700 AS2/LOB AT4G30980 HLH/MYC AT4G31650 REM AT5G62430 Z-Dof AT4G00220 AS2/LOB AT4G16430 HLH/MYC AT4G34400 REM AT1G47655 Z-Dof AT4G00210 AS2/LOB AT1G63650 HLH/MYC AT3G18990 REM AT5G66940 Z-Dof AT1G65620 AS2/LOB AT5G53210 HLH/MYC AT3G17010 REM AT5G65590 Z-Dof AT1G07900 AS2/LOB AT3G61950 HLH/MYC AT3G06160 REM AT1G26790 Z-Dof AT1G16530 AS2/LOB AT2G46810 HLH/MYC AT2G35310 REM AT4G21080 Z-Dof AT1G31320 AS2/LOB AT4G01460 HLH/MYC AT2G16210 REM AT2G28510 Z-Dof AT1G36000 AS2/LOB AT5G46690 HLH/MYC AT4G31640 REM AT4G38000 Z-Dof AT2G45430 AT-Hook AT1G09530 HLH/MYC AT4G31630 REM AT2G46590 Z-Dof AT3G18035 AT-Hook AT4G21330 HLH/MYC AT4G31615 REM AT5G02460 Z-Dof AT3G60870 AT-Hook AT2G41130 HLH/MYC AT4G31620 REM AT1G21340 Z-Dof AT5G49700 AT-Hook AT5G64340 HLH/MYC AT4G31610 REM AT3G45610 Z-Dof AT4G12080 AT-Hook AT5G46760 HLH/MYC AT2G37120 S1FA AT4G21040 Z-Dof AT3G55560 AT-Hook AT2G16910 HLH/MYC AT3G09735 S1FA AT1G28310 Z-Dof AT1G76500 AT-Hook AT4G09180 HLH/MYC AT3G53370 S1FA AT4G21030 Z-Dof AT1G14490 AT-Hook AT5G09460 HLH/MYC AT5G43270 SBP AT4G21050 Z-Dof AT3G04590 AT-Hook AT2G27230 HLH/MYC AT2G42200 SBP AT1G29160 Z-Dof AT3G04570 AT-Hook AT2G34820 HLH/MYC AT3G15270 SBP AT5G62940 Z-Dof AT1G63470 AT-Hook AT4G33880 HLH/MYC AT2G47070 SBP AT5G39660 Z-Dof AT1G63480 AT-Hook AT4G34530 HLH/MYC AT5G50670 SBP AT3G61850 Z-Dof AT2G45850 AT-Hook AT5G67060 HLH/MYC AT1G27370 SBP AT4G24060 Z-Dof AT4G22810 AT-Hook AT3G26744 HLH/MYC AT1G20980 SBP AT1G64620 Z-Dof AT4G14465 AT-Hook AT2G24260 HLH/MYC AT1G53160 SBP AT3G47500 Z-Dof AT4G17950 AT-Hook AT5G46830 HLH/MYC AT1G76580 SBP AT1G01780 Z-LIM AT1G20900 AT-Hook AT5G09750 HLH/MYC AT3G60030 SBP AT1G10200 Z-LIM AT4G25320 AT-Hook AT2G20180 HLH/MYC AT5G18830 SBP AT2G45800 Z-LIM AT1G48620 AT-Hook AT2G18300 HLH/MYC AT3G57920 SBP AT3G61230 Z-LIM AT5G62260 AT-Hook AT2G43010 HLH/MYC AT1G02065 SBP AT2G39900 Z-LIM AT2G42940 AT-Hook AT4G09820 HLH/MYC AT1G27360 SBP AT3G55770 Z-LIM AT5G51590 AT-Hook AT2G46510 HLH/MYC AT2G33810 SBP AT1G02170 Z-LSDlike AT5G46640 AT-Hook AT1G12860 HLH/MYC AT1G69170 SBP AT5G64240 Z-LSDlike AT4G17800 AT-Hook AT2G22760 HLH/MYC AT2G29060 SCR AT4G25110 Z-LSDlike AT2G35270 AT-Hook AT1G09250 HLH/MYC AT1G55580 SCR AT4G21610 Z-LSDlike AT4G35390 AT-Hook AT2G14760 HLH/MYC AT5G41920 SCR AT1G32540 Z-LSDlike AT4G00200 AT-Hook AT1G10120 HLH/MYC AT1G07520 SCR AT4G20380 Z-LSDlike AT3G61310 AT-Hook AT2G22750 HLH/MYC AT2G04890 SCR AT2G41590 Z-Tall-1 AT4G12050 AT-Hook AT4G21340 HLH/MYC AT4G37650 SCR AT2G36930 Z-ZPF AT1G22310 AT-Hook AT4G28790 HLH/MYC AT3G46600 SCR AT5G22480 Z-ZPF AT1G14900 AT-Hook AT2G46970 HLH/MYC AT5G48150 SCR AT3G28920 ZF-HB AT4G22770 AT-Hook AT1G22490 HLH/MYC AT3G50650 SCR AT5G15210 ZF-HB AT1G48610 AT-Hook AT3G24140 HLH/MYC AT3G03450 SCR AT1G14440 ZF-HB AT2G33620 AT-Hook AT4G28800 HLH/MYC AT3G49950 SCR AT2G02540 ZF-HB AT3G50750 BES AT2G42300 HLH/MYC AT2G45160 SCR AT1G69600 ZF-HB AT4G18890 BES AT3G21330 HLH/MYC AT1G50600 SCR AT5G39760 ZF-HB AT1G78700 BES AT1G06150 HLH/MYC AT3G60630 SCR AT5G60480 ZF-HB AT1G19350 BES AT1G35460 HLH/MYC AT4G08250 SCR AT1G14687 ZF-HB AT1G75080 BES AT1G64625 HLH/MYC AT3G13840 SCR AT5G42780 ZF-HB AT4G36780 BES AT2G31280 HLH/MYC AT1G07530 SCR AT3G50890 ZF-HB AT3G12560 BPF-1 AT2G28160 HLH/MYC AT5G17490 SCR AT2G18350 ZF-HB AT5G59430 BPF-1 AT5G62610 HLH/MYC AT1G66350 SCR AT1G75240 ZF-HB AT3G53790 BPF-1 AT5G01310 HLH/MYC AT1G14920 SCR AT4G24660 ZF-HB AT1G07540 BPF-1 AT3G07340 HLH/MYC AT2G01570 SCR AT5G65410 ZF-HB AT3G46590 BPF-1 AT3G56770 HLH/MYC AT3G54220 SCR AT5G24800 bZIP AT5G13820 BPF-1 AT1G72210 HLH/MYC AT4G36710 SCR AT5G28770 bZIP AT5G12840 CAAT AT5G10570 HLH/MYC AT5G67411 SCR AT1G77920 bZIP AT1G17590 CAAT AT4G23800 HMG AT4G17230 SCR AT5G15830 bZIP AT2G34720 CAAT AT4G35570 HMG AT5G66770 SCR AT4G34000 bZIP AT3G05690 CAAT AT3G51880 HMG AT4G00150 SCR AT5G38800 bZIP AT3G48590 CAAT AT5G23420 HMG AT2G37650 SCR AT5G44080 bZIP AT1G54830 CAAT AT1G20693 HMG AT5G52510 SCR AT5G06839 bZIP AT1G72830 CAAT AT2G17560 HMG AT5G59450 SCR AT1G68640 bZIP AT5G43250 CAAT AT3G28730 HMG AT1G63100 SCR AT2G36270 bZIP AT2G47810 CAAT AT1G20696 HMG AT1G21450 SCR AT3G62420 bZIP AT5G63470 CAAT AT4G11080 HMG AT1G50420 SCR AT2G24340 bZIP AT1G54160 CAAT AT2G34450 HMG AT5G66350 SRS AT1G13600 bZIP AT2G13570 CAAT AT5G16820 HS AT3G51060 SRS AT5G08139 bZIP AT5G27910 CAAT AT3G02990 HS AT5G33210 SRS AT3G44460 bZIP AT5G47670 CAAT AT3G51910 HS AT2G18120 SRS AT3G56660 bZIP AT5G50480 CAAT AT4G17600 HS AT1G75520 SRS AT2G40950 bZIP AT5G50470 CAAT AT3G22830 HS AT1G19790 SRS AT2G41070 bZIP AT5G50490 CAAT AT5G43840 HS AT4G36260 SRS AT3G10800 bZIP AT5G38140 CAAT AT1G32330 HS AT2G21400 SRS AT2G04038 bZIP AT2G37060 CAAT AT5G03720 HS AT5G12330 SRS AT3G58120 bZIP AT3G14020 CAAT AT3G63350 HS AT3G54430 SRS AT2G42380 bZIP AT5G06510 CAAT AT2G41690 HS AT4G00390 STK AT2G21230 bZIP AT1G08970 CAAT AT2G26150 HS AT1G11510 STK AT3G30530 bZIP AT1G09030 CAAT AT5G45710 HS AT1G61730 STK AT1G42990 bZIP AT4G14540 CAAT AT4G13980 HS AT4G00250 STK AT2G22850 bZIP AT5G23090 CAAT AT4G36990 HS AT4G00610 STK AT3G54620 bZIP AT1G56170 CAAT AT4G17750 HS AT4G01260 STK AT1G08320 bZIP AT5G47640 CAAT AT4G18880 HS AT3G04930 STK AT3G56850 bZIP AT2G38880 CAAT AT4G18870 HS AT5G14280 STK AT3G19290 bZIP AT1G21970 CAAT AT5G62020 HS AT5G28040 STK AT2G35530 bZIP AT3G53340 CAAT AT4G11660 HS AT4G00270 STK AT5G60830 bZIP AT1G30500 CAAT AT5G54070 HS AT2G36340 STK AT3G51960 bZIP AT2G27470 CAAT AT1G67970 HS AT2G01370 STK AT1G75390 bZIP AT3G20910 CAAT AT1G77570 HS AT2G25650 STK AT5G49450 bZIP AT5G08190 CAAT AT1G46264 HS AT4G25210 STK AT2G16770 bZIP AT1G07980 CAAT AT4G19630 HS AT1G66420 STK AT3G17609 bZIP AT3G12480 CAAT AT3G24520 HS AT1G44810 STK AT4G37730 bZIP AT3G12890 CCT AT5G25890 IAA AT1G50410 SWI/SNF AT4G38900 bZIP AT2G32310 CCT AT3G23030 IAA AT5G20420 SWI/SNF AT2G40620 bZIP AT1G07050 CCT AT1G80390 IAA AT5G19310 SWI/SNF AT4G34590 bZIP AT5G59990 CCT AT1G04550 IAA AT1G11100 SWI/SNF AT1G03970 bZIP AT4G27900 CCT AT2G33310 IAA AT1G02670 SWI/SNF AT2G46270 bZIP AT5G53420 CCT AT3G15540 IAA AT1G05120 SWI/SNF AT4G01120 bZIP AT1G63820 CCT AT1G52830 IAA AT1G08060 SWI/SNF AT4G36730 bZIP AT5G41380 CCT AT2G22670 IAA AT5G22750 SWI/SNF AT2G31370 bZIP AT5G14370 CCT AT1G15580 IAA AT1G08600 SWI/SNF AT5G06950 bZIP AT4G25990 CCT AT5G65670 IAA AT2G16390 SWI/SNF AT3G49760 bZIP AT1G04500 CCT AT3G04730 IAA AT2G21450 SWI/SNF AT1G68880 bZIP AT5G57180 CCT AT3G23050 IAA AT3G24340 SWI/SNF AT1G06850 bZIP AT2G33350 CCT AT1G15050 IAA AT3G06010 SWI/SNF AT2G12900 bZIP AT4G14770 CPP AT4G32280 IAA AT1G03750 SWI/SNF AT2G12940 bZIP AT3G04850 CPP AT5G57420 IAA AT5G66750 SWI/SNF AT2G13150 bZIP AT5G25790 CPP AT2G01200 IAA AT2G44980 SWI/SNF AT1G35490 bZIP AT4G29000 CPP AT2G46990 IAA AT2G18760 SWI/SNF AT1G49720 bZIP AT3G22780 CPP AT1G51950 IAA AT1G48310 SWI/SNF AT1G59530 bZIP AT3G22760 CPP AT4G28640 IAA AT5G63950 SWI/SNF AT1G43700 bZIP AT3G16160 CPP AT4G14560 IAA AT3G19210 SWI/SNF AT2G21235 bZIP AT2G20110 CPP AT3G16500 IAA AT3G54460 SWI/SNF AT5G42910 bZIP AT4G32780 DBP AT3G17600 IAA AT1G61140 SWI/SNF AT1G45249 bZIP AT4G14740 DBP AT4G29080 IAA AT3G06400 SWI/SNF AT1G32150 bZIP AT4G17410 DBP AT3G62100 IAA AT5G18620 SWI/SNF AT4G35900 bZIP AT1G45207 DBP AT5G43700 IAA AT5G43530 SWI/SNF AT4G02640 bZIP AT2G45820 DBP AT1G04250 IAA AT2G40770 SWI/SNF AT5G11260 bZIP AT4G36970 DBP AT1G04240 IAA AT5G22750 SWI/SNF AT5G10030 bZIP AT3G63300 DBP AT4G14550 IAA AT5G05130 SWI/SNF AT5G06960 bZIP AT3G48940 DBP AT1G04100 IAA AT3G16600 SWI/SNF AT5G65210 bZIP AT1G30320 DBP AT2G34880 JUMONJI AT3G12810 SWI/SNF AT1G22070 bZIP AT3G57540 DBP AT5G46910 JUMONJI AT1G05490 SWI/SNF AT2G17770 bZIP AT5G57770 DBP AT3G48430 JUMONJI AT4G31900 SWI/SNF AT5G04840 bZIP AT5G43870 DBP AT5G04240 JUMONJI AT2G13370 SWI/SNF AT3G12250 bZIP AT5G47430 DBP AT1G63490 JUMONJI AT2G28290 SWI/SNF AT4G35040 bZIP AT2G41870 DBP AT2G38950 JUMONJI AT5G44800 SWI/SNF AT1G06070 bZIP AT3G61260 DBP AT1G08620 JUMONJI AT3G54280 SWI/SNF AT2G18160 bZIP AT5G23750 DBP AT1G30810 JUMONJI AT3G20010 SWI/SNF AT3G59580 bZIP-NIN AT4G17350 DBP AT4G20400 JUMONJI AT3G42670 SWI/SNF AT2G17150 bZIP-NIN AT2G02170 DBP AT5G24890 KCL AT2G25170 SWI/SNF AT1G64530 bZIP-NIN AT4G00670 DBP AT4G31510 KCL AT3G57300 SWI/SNF AT2G43500 bZIP-NIN AT1G67590 DBP AT2G24550 KCL AT2G02090 SWI/SNF AT1G20640 bZIP-NIN AT4G16670 DBP AT1G31140 MADS AT2G46020 SWI/SNF AT1G18790 bZIP-NIN AT3G22810 DBP AT3G57390 MADS AT1G67260 TEO AT1G74480 bZIP-NIN AT2G33700 DBPP AT3G57230 MADS AT1G30210 TEO AT5G66990 bZIP-NIN AT3G62260 DBPP AT4G22950 MADS AT4G18390 TEO AT5G53040 bZIP-NIN AT1G48040 DBPP AT5G38620 MADS AT3G02150 TEO AT4G35590 bZIP-NIN AT3G51470 DBPP AT1G46408 MADS AT2G31070 TEO AT1G76350 bZIP-NIN AT3G17250 DBPP AT1G69540 MADS AT3G15030 TEO AT4G24020 bZIP-NIN AT2G25620 DBPP AT1G18750 MADS AT1G68800 TEO AT4G38340 bZIP-NIN AT3G48160 E2F AT1G22590 MADS AT5G60970 TEO AT4G35270 bZIP-NIN AT5G14960 E2F AT5G27960 MADS AT3G18550 TEO AT1G09950 bZIP-ZW2 AT5G02470 E2F AT2G28700 MADS AT5G08070 TEO AT4G18660 bZIP-ZW2 AT2G36010 E2F AT1G28460 MADS AT1G53230 TEO AT4G18690 bZIP-ZW2 AT1G47870 E2F AT1G28450 MADS AT1G13450 TH AT1G58330 bZIP-ZW2 AT5G22220 E2F AT2G40210 MADS AT1G76880 TH AT3G14880 bZIP-ZW2 AT5G03415 E2F AT3G04100 MADS AT5G47660 TH AT5G45830 bZIP-ZW2 AT3G01330 E2F AT1G72350 MADS AT4G17050 TH AT4G18680 bZIP-ZW2 AT2G27050 EIL AT5G23260 MADS AT3G58630 TH AT4G18650 bZIP-ZW2 AT5G21120 EIL AT5G60910 MADS AT4G31270 TH AT1G77500 bZIPt2 AT5G10120 EIL AT2G45660 MADS AT2G33550 TH AT3G60320 bZIPt2 AT3G20770 EIL AT5G26650 MADS AT2G38250 TH AT2G27090 bZIPt2 AT5G65100 EIL AT5G26630 MADS AT5G28300 TH AT4G39790 bZIPt2 AT1G73730 EIL AT5G26580 MADS AT5G03680 TH AT1G52320 bZIPt2 AT1G09060 ENBP AT2G26320 MADS AT5G63420 TH AT1G20530 bZIPt2 AT1G62310 ENBP AT1G65300 MADS AT2G35640 TH AT2G19090 bZIPt2 AT4G21430 ENBP AT4G37940 MADS AT3G25990 TH AT4G35240 bZIPt2 AT4G00990 ENBP AT1G71692 MADS AT1G31310 TH AT2G34670 bZIPt2 AT1G11950 ENBP AT4G09960 MADS AT3G10040 TH AT3G51290 bZIPt2 AT3G07610 ENBP AT1G24260 MADS AT3G24860 TH AT2G17110 bZIPt2 AT4G21670 FRY AT5G49490 MADS AT5G05550 TH AT5G54480 bZIPt2 AT5G01270 FRY AT5G49420 MADS AT3G54390 TH AT5G25590 bZIPt2 AT2G38300 GARP AT2G45650 MADS AT3G11100 TH AT1G02110 bZIPt2 AT5G44190 GARP AT2G14210 MADS AT3G14180 TH AT5G62320 MYB- AT1G79430 GARP AT2G22630 MADS AT5G01380 TH (R1)R2R3 AT4G01680 MYB- AT1G66370 MYB- AT5G55020 MYB- AT5G11050 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 (R1)R2R3 AT3G30210 MYB- AT1G66380 MYB- AT2G26960 MYB- AT5G02840 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT4G00540 MYB- AT5G67300 MYB- AT3G28470 MYB- AT5G56840 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT4G38620 MYB- AT5G57620 MYB- AT4G37780 MYB- AT1G19000 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G66390 MYB- AT5G59780 MYB- AT5G52600 MYB- AT5G23650 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT5G10280 MYB- AT5G40350 MYB- AT4G13480 MYB- AT5G37260 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT2G37630 MYB- AT2G47460 MYB- AT5G49620 MYB- AT5G01200 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G79180 MYB- AT4G37260 MYB- AT1G56650 MYB- AT4G01280 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G22640 MYB- AT2G47190 MYB- AT4G05100 MYB- AT1G17520 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT4G32730 MYB- AT4G25560 MYB- AT1G56160 MYB- AT1G19510 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT5G60890 MYB- AT2G02820 MYB- AT1G26780 MYB- AT1G75250 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT5G58850 MYB- AT1G63910 MYB- AT2G13960 MYB- AT1G01380 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT3G08500 MYB- AT5G02320 MYB- AT3G18100 MYB- AT1G49950 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G25340 MYB- AT3G02940 MYB- AT3G13890 MYB- AT3G49850 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G18710 MYB- AT1G74080 MYB- AT5G49330 MYB- AT3G10580 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT3G62610 MYB- AT3G13540 MYB- AT3G46130 MYB- AT4G36570 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G66230 MYB- AT5G15310 MYB- AT1G43330 MYB- AT5G08520 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT5G14750 MYB- AT4G09460 MYB- AT4G18770 MYB- AT5G61620 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G34670 MYB- AT3G50060 MYB- AT4G26930 MYB- AT5G04760 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT3G48920 MYB- AT5G26660 MYB- AT2G25230 MYB- AT5G17300 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G18570 MYB- AT3G27810 MYB- AT3G29020 MYB- AT5G05790 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G09540 MYB- AT4G17785 MYB- AT4G21440 MYB- AT5G58900 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT5G35550 MYB- AT3G09230 MYB- AT4G34990 MYB- AT2G38090 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT5G62470 MYB- AT2G39880 MYB- AT1G06180 MYB- AT2G21650 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT5G16600 MYB- AT4G33450 MYB- AT5G65230 MYB- AT4G09450 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT3G28910 MYB- AT3G11440 MYB- AT5G17800 MYB- AT5G53200 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT3G47600 MYB- AT1G68320 MYB- AT5G61420 MYB- AT5G52660 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G18960 MYB- AT1G16490 MYB- AT5G07690 MYB- AT4G39250 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT3G12720 MYB- AT3G01530 MYB- AT4G12350 MYB- AT2G46830 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT2G32460 MYB- AT1G73410 MYB- AT1G35515 MYB- AT1G01520 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G74650 MYB- AT1G17950 MYB- AT1G08810 MYB- AT2G30420 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT2G16720 MYB- AT1G57560 MYB- AT3G24310 MYB- AT2G46410 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G48000 MYB- AT5G12870 MYB- AT3G27920 MYB- AT3G09600 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT5G11510 MYB- AT4G28110 MYB- AT3G49690 MYB- AT1G49010 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT3G60460 MYB- AT5G14340 MYB- AT3G23250 MYB- AT1G18330 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT3G55730 MYB- AT2G36890 MYB- AT5G65790 MYB- AT3G10590 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G14350 MYB- AT5G23000 MYB- AT4G22680 MYB- AT1G72740 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT5G54230 MYB- AT5G06100 MYB- AT2G23290 MYB- AT2G30432 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT5G56110 MYB- AT3G53200 MYB- AT5G40430 MYB- AT5G67580 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT5G07700 MYB- AT5G52260 MYB- AT5G40330 MYB- AT1G71030 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT3G09370 MYB- AT3G61250 MYB- AT5G39700 MYB- AT1G74840 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT3G01140 MYB- AT2G31180 MYB- AT3G27785 MYB- AT1G70000 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G74430 MYB- AT3G12820 MYB- AT5G40360 MYB- AT1G09770 MYB- (R1)R2R3 (R1)R2R3 (R1)R2R3 related AT1G69560 MYB- AT5G16770 MYB- AT2G18328 MYB-related AT3G16350 MYB- (R1)R2R3 (R1)R2R3 related AT3G06490 MYB- AT2G26950 MYB- AT5G47390 MYB-related AT4G01060 MYB- (R1)R2R3 (R1)R2R3 related AT3G11280 MYB- related AT1G01060 MYB- related 

What is claimed is:
 1. A synthetic chimeric polypeptide comprising a transcription activation domain of: (i) SEQ ID NO: 55; (ii) SEQ ID NO: 56; (iii) SEQ ID NO: 94; (iv) SEQ ID NO: 95; (v) an amino acid sequence with a minimum percentage identity to any of SEQ ID NO: 37-54, wherein the minimum percentage identity is selected from the group consisting of 100%, 95%, 93.8%, 90%, 87.5%, 85%, 81.2%, 80%, 75.0%, 70%, 68.7%, 65%, 62.5%, 60%, 56.2%, 50%, and 43.7%; (vi) an amino acid sequence encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 57-63 and 76-93; or (vii) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of SEQ ID NO: 57-63 or 76-93, wherein the stringent conditions comprise at least 6×SSC and 1% SDS at 65° C. with a first wash step for 10 minutes at about 42° C. with about 20% (w/v) formamide in 0.1×SSC and with a subsequent wash step for 10 minutes with 0.2×SSC and 0.1% SDS at 65° C.; wherein the transcription activation domain is covalently fused to a transcription regulatory polypeptide; and wherein the transcription activation domain and the transcription regulatory polypeptide are mutually heterologous and do not occur in nature in the same protein.
 2. The chimeric polypeptide of claim 1, wherein the transcription regulatory polypeptide is a transcription factor polypeptide.
 3. The chimeric polypeptide of claim 1, wherein the chimeric polypeptide comprises any of SEQ ID NOs: 37-54 or SEQ ID NO:
 56. 4. The chimeric polypeptide of claim 1, wherein expression of the chimeric polypeptide is regulated by an inducible, developmental or tissue-specific promoter.
 5. A recombinant polynucleotide encoding the chimeric polypeptide according to claim
 1. 6. A host plant cell comprising a nucleic acid construct encoding a chimeric polypeptide comprising a transcription activation domain covalently fused to a transcription regulatory polypeptide, wherein the transcription activation domain and the transcription regulatory polypeptide are mutually heterologous and do not occur in nature in the same protein, or do not occur in the same copy number or configuration in nature; and wherein the transcription activation domain comprises: (i) SEQ ID NO: 55; (ii) SEQ ID NO: 56; (iii) SEQ ID NO: 94; (iv) SEQ ID NO: 95; (v) an amino acid sequence with a minimum percentage identity to any of SEQ ID NO: 37-54, wherein the minimum percentage identity is selected from the group consisting of 100%, 95%, 93.8%, 90%, 87.5%, 85%, 81.2%, 80%, 75.0%, 70%, 68.7%, 65%, 62.5%, 60%, 56.2%, 50%, and 43.7%; (vi) an amino acid sequence encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 57-63 and 76-93; or (vii) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of SEQ ID NO: 57-63 or 76-93, wherein the stringent conditions comprise at least 6×SSC and 1% SDS at 65° C. with a first wash step for 10 minutes at about 42° C. with about 20% (w/v) formamide in 0.1×SSC and with a subsequent wash step for 10 minutes with 0.2×SSC and 0.1% SDS at 65° C.
 7. The host cell of claim 6, wherein the transcription regulatory polypeptide is a transcription factor polypeptide.
 8. The host cell of claim 6, wherein the chimeric polypeptide comprises any of SEQ ID NOs: 37-54 or SEQ ID NO:
 56. 9. The host cell of claim 6, wherein expression of the chimeric polypeptide is regulated by an inducible, developmental or tissue-specific promoter.
 10. A transgenic plant comprising a host plant cell according to claim
 6. 11. A method for increasing the expression of a target polynucleotide sequence, the methods steps comprising: (a) generating a nucleic acid construct encoding a chimeric polypeptide comprising a transcription activation domain covalently fused to a transcription regulatory polypeptide, wherein the transcription activation domain and the transcription regulatory polypeptide are mutually heterologous and do not occur in nature in the same protein, or do not occur in the same copy number or configuration in nature; wherein the chimeric polypeptide activates transcription of the target polynucleotide sequence to which the chimeric polypeptide binds, and wherein the transcription activation domain comprises: (i) SEQ ID NO: 55; (ii) SEQ ID NO: 56; (iii) SEQ ID NO: 94; (iv) SEQ ID NO: 95; (v) an amino acid sequence with a minimum percentage identity to any of SEQ ID NO: 37-54, wherein the minimum percentage identity is selected from the group consisting of 100%, 95%, 93.8%, 90%, 87.5%, 85%, 81.2%, 80%, 75.0%, 70%, 68.7%, 65%, 62.5%, 60%, 56.2%, 50%, and 43.7%; (vi) an amino acid sequence encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 57-63 and 76-93; or (vii) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of SEQ ID NO: 57-63 or 76-93, wherein the stringent conditions comprise at least 6×SSC and 1% SDS at 65° C. with a first wash step for 10 minutes at about 42° C. with about 20% (w/v) formamide in 0.1×SSC and with a subsequent wash step for 10 minutes with 0.2×SSC and 0.1% SDS at 65° C.; and (b) introducing the nucleic acid construct into a host cell.
 12. The method of claim 11, wherein the transcription regulatory polypeptide is a transcription factor polypeptide.
 13. The method of claim 11, wherein the chimeric polypeptide comprises any of SEQ ID NOs: 37-54 or SEQ ID NO:
 56. 14. The method of claim 11, wherein expression of the chimeric polypeptide is regulated by an inducible, developmental or tissue-specific promoter. 